What Are Apoptosis Pathways?

Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain internal environment stability. Apoptosis is different from cell necrosis. Apoptosis is not a passive process but an active process. It involves the activation, expression and regulation of a series of genes. It is not a kind of autologous injury under pathological conditions. Phenomenon, but a process of death actively striving for better adaptation to the living environment.

Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain internal environment stability. Apoptosis is different from cell necrosis. Apoptosis is not a passive process but an active process. It involves the activation, expression and regulation of a series of genes. It is not a kind of autologous injury under pathological conditions. Phenomenon, but a process of death actively striving for better adaptation to the living environment.

Chinese name

Apoptosis

Foreign name

Apoptosis

Apoptosis

Cells in the human body are destined to die. Some deaths are physiological and some deaths are pathological. Research on the process of cell death has become a hot spot in biological and medical research. It has been known that there are at least two ways of cell death, namely cell necrosis and apoptosis. Cell necrosis is a cell death method that has long been recognized, while apoptosis is a cell death method that has been gradually recognized.

Apoptosis is a basic biological phenomenon of cells and plays a necessary role in removing unwanted or abnormal cells in multicellular organisms. It plays an important role in the evolution of organisms, the stabilization of the internal environment, and the development of multiple systems. Apoptosis is not only a special type of cell death, but also has important biological significance and complex molecular biological mechanisms.

Apoptosis is a strictly controlled process by multiple genes. These genes are very conserved among species, such as the Bcl-2 family, caspase family, oncogenes such as C-myc, and tumor suppressor gene P53, etc. With the development of molecular biology technology, a variety of apoptosis processes have been developed. It is quite well understood, but the exact mechanism of the apoptotic process is not completely clear so far. The disorder of the apoptotic process may be directly or indirectly related to the occurrence of many diseases. Such as tumors, autoimmune diseases, etc., there are many factors that can induce apoptosis, such as radiation and drugs.

Part of the human physiological structure belongs to natural apoptosis, such as the tailed stage of human, the tail automatically apoptotic during development.

Apoptotic process

Apoptosis Research History

1. Formation of the concept of apoptosis. In 1965, Australian scientists discovered that after ligating the portal vein of rats, some dead cells were scattered in the liver parenchyma under electron microscope. The lysosomes of these cells were not destroyed, which is obviously different from cell necrosis. These cells shrink in volume, agglutinate chromatin, fall off from their surrounding tissues and be swallowed, and the body has no inflammatory response. Kerr and other three scientists first proposed the concept of apoptosis in 1972, announcing the beginning of the true exploration of apoptosis. Prior to this, studies on embryonic developmental biology, immune system, and liver cell death were all The foundation of this concept was laid.

2. Stage of morphology and biochemistry of apoptosis (1972-1987).

1) The morphological characteristics were studied in detail using light and electron microscopy.

2) Degradation of chromosomal DNA: A significant feature of apoptosis is the degradation of chromatin DNA. The size of DNA fragments during apoptosis is an integer multiple of 200bp.

3) Synthesis of RNA / protein macromolecules.

4) The change of calcium ion, the increase of intracellular calcium ion concentration is an important condition for cell apoptosis.

5) Endogenous endonuclease: This kind of endonuclease is required for cell apoptosis.

3 Molecular biology research phase of apoptosis.

1) Related genes and regulation of apoptosis.

2) Signal transduction of apoptosis.

3) Various molecules and their interactions and relationships with apoptosis.

4 The clinical application of apoptosis in the basic research stage of apoptosis research, its vitality lies in the ultimate benefit of the clarification of disease mechanisms, and the exploration and development of new therapies.

Apoptosis concept explained

Apoptosis and programmed cell death

In fact, in a strict lexical sense, programmed cell death (PCD) is very different from apoptosis. The concept of programmed cell death was introduced in 1956. PCD is a functional concept, describing that certain cell death in a multicellular organism is a normal part of the individual's development that is strictly controlled by strict procedures. For example, tadpoles become frogs. The disappearance of the tail during metamorphosis is accompanied by a large number of cell deaths. The disappearance of interfin webs, jaw fusion, retinal development, and normal development of the immune system in higher mammals must involve cell death. These various cell deaths that occur during the development of the body have a common feature: that is, they die and disappear from the normal tissue one by one, the body has no inflammatory response, and it is beneficial and necessary for the development of the entire body. Therefore, it is considered that the programmed cell death during animal development is a developmental concept, while apoptosis is a morphological concept, describing a completely different form of cell death from necrosis with a set of morphological characteristics. However, it is generally believed that the two concepts of apoptosis and programmed death can be used interchangeably and have the same meaning.

Development of fetal hands

The difference between apoptosis and necrosis

Although the final results of apoptosis and necrosis are very similar, their process and performance are very different.

Necrosis: Necrosis is a death process in which cells are subject to strong physical and chemical or biological factors that cause the cells to change disorderly. It is characterized by cell swelling, cell membrane rupture, cell contents overflow, slow nuclear changes, insufficient DNA degradation, and causing severe local inflammation.

Apoptosis is a death process in which cells respond to the physiological and pathological stimuli of the environment, changes in environmental conditions, or orderly changes in mitigation damage. The changes in cells and tissues are significantly different from necrosis.

process

1.Initiation of apoptosis

2.Apoptotic body formation

3. Apoptotic bodies are gradually engulfed by neighboring cells or phagocytic cells in vivo, and the residual material of apoptotic cells is digested and reused.

Morphological changes of apoptosis

Morphological observation of changes in apoptosis is multi-stage. Apoptosis often involves a single cell, and even a small number of cells occur asynchronously. The first appears that the cell volume shrinks, the connection disappears, and it detaches from the surrounding cells, then the cytoplasm density increases, the mitochondrial membrane potential disappears, the permeability changes, cytochrome C is released into the cytoplasm, the cytoplasm is concentrated, and the nuclear membrane nucleoli is broken. DNA is degraded into fragments of about 180bp-200bp; vesicles are formed on the cell membrane, and the phosphatidylserine inside the membrane is turned out to the membrane surface. The membrane structure is still intact, and the apoptotic cell remains can be divided into several apoptotic cells. Body, there is no content overflow, so it does not cause surrounding inflammatory response, apoptotic bodies can be quickly engulfed by surrounding full-time or non-full-time phagocytic cells.

Apoptotic biochemical changes

1) Fragmentation of DNA

A significant feature of apoptosis is the DNA degradation of the cell chromosomes, which is a more common phenomenon.

Apoptosis

This degradation is very specific and regular. The resulting DNA fragments of different lengths are about multiples of 180-200bp, and this is exactly the length of the histone oligomers, suggesting that the chromosomal DNA happens to be between the nucleosome and the nucleus. The junction of the corpuscle was cut off, resulting in oligonucleosome fragments of different lengths. Experiments have shown that the controlled degradation of this DNA is the result of the action of an endogenous endonuclease, which is linked to the nucleosome. The chromosomal DNA was cut off at the site, and this degradation showed a specific ladder-like Ladder pattern in agarose gel electrophoresis, while necrosis showed a diffuse continuous pattern.

2) Macromolecular Synthesis

The biochemical changes of apoptosis are not only the controlled degradation of DNA, but also the expression of new genes and the synthesis of certain biological macromolecules as regulatory factors in the process of apoptosis. For example, TFAR-19 found in our laboratory is a molecule that is highly expressed during apoptosis. For example, in the process of glucocorticoid-induced mouse thymocyte apoptosis, RNA synthesis inhibitors or protein synthesis inhibitors can be added to inhibit cells. The occurrence of apoptosis.

Mechanism of apoptosis process

The process of apoptosis can be roughly divided into the following stages:

Accept apoptosis signal Interaction between apoptosis regulatory molecules Proteolytic enzyme activation (Caspase) Enter a continuous reaction process

Apoptotic initiation stage

The initiation of apoptosis is the opening or closing of a series of control switches inside the cell after the corresponding signal is stimulated. Different external factors initiate apoptosis in different ways, and the signal transduction caused by them is also different. Objectively speaking, The understanding of the signal transmission system in the process of apoptosis is still not comprehensive. The clearer pathways are mainly:

1) Membrane receptor pathway of apoptosis: Various external factors are initiators of apoptosis. They can transmit apoptosis signals through different signaling systems and cause apoptosis. We take Fas-FasL as an example:

Fas is a transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. Combining FasL with FasL can initiate the transduction of apoptosis signals and cause apoptosis. Its activation involves a series of steps: first, the ligand induces receptor trimerization, and then forms an apoptosis-inducing complex on the cell membrane, which includes a Fas-related protein FADD with a death domain. Fas, also known as CD95, is a receptor molecule composed of 325 amino acids. Once Fas binds to its ligand FasL, Fas molecules can start lethal signal transduction, and eventually cause a series of characteristic changes in the cell, causing the cell to die. Fas, as a commonly expressed receptor molecule, can appear on the surface of many cells, but the expression of FasL has its own characteristics. It usually only appears on activated T cells and NK cells, so it has been activated by killer immune cells. It is often the most effective way to target cells to death by the apoptotic pathway. The intracellular segment of the Fas molecule carries a special death domain (DD). After the trimeric Fas and FasL are combined, the death domains of the three Fas molecules are clustered into a cluster, which attracts another protein, FADD, with the same death domain in the cytoplasm. FADD is a connexin in the death signal transcription. It consists of two parts: the C-terminal (DD domain) and N-terminal (DED) parts. The DD domain is responsible for binding to the DD domain on the intracellular segment of the Fas molecule, and the protein is connected to another subsequent component with DED by DED, thereby causing the N-segment DED to immediately interact with the inactive cysteine protease 8 ( caspase8) zymogen cross-links homophilically, polymerizing multiple caspase8 molecules, the caspase8 molecule is converted from a single-chain zymogen to an active double-chain protein, which then causes a subsequent cascade reaction, namely Caspases, which is used as It is activated, causing the following cascade. Cells undergo apoptosis. Therefore, the TNF-induced apoptosis pathway is similar to this.

2) Biochemical pathways for cytochrome C release and Caspases activation

Mitochondria is the control center of cell life activity. It is not only the center of cellular respiratory chain and oxidative phosphorylation, but also the center of apoptosis regulation. Experiments have shown that the release of cytochrome C from mitochondria is a key step in apoptosis. In the presence of dATP, cytochrome C released into the cytoplasm can bind to apoptosis-associated factor 1 (Apaf-1) to form multimers, and promote caspase-9 to form apoptotic bodies, 9 is activated, activated caspase-9 can activate other caspases such as caspase-3, etc., thereby inducing apoptosis. In addition, mitochondria also release apoptosis-inducing factors, such as AIF, which are involved in activating caspase. It can be seen that the relevant components of apoptotic bodies exist in different parts of normal cells. Pro-apoptotic factors can induce the release of cytochrome C and the formation of apoptotic bodies. Obviously, the regulation of cytochrome C release from mitochondria is a key issue in the molecular mechanism of apoptosis. Most apoptosis-stimulating factors activate the apoptotic pathway through mitochondria. Some people think that the receptor-mediated apoptosis pathway also has the release of cytochrome C from mitochondria. For example, in a cell that responds to Fas, a type of cell (type1) contains sufficient caspase 8 that can be activated by the death receptor and cause apoptosis. Overexpression of Bcl-2 in such cells does not inhibit Fas-induced apoptosis. In another type of cell (type 2) such as liver cells, Fas receptor-mediated activation of cystase 8 cannot reach very high levels. Therefore, the apoptotic signal in such cells needs to be amplified by the mitochondrial pathway of apoptosis. Bid, a Bcl-2 family protein containing only the BH3 domain, transmits the apoptotic signal from cystase 8 to mitochondria. messenger.

Apoptosis execution

Although the detailed mechanism of the apoptotic process is not completely clear, it has been determined that Caspase, caspase, plays an essential role in the process of apoptosis. The process of apoptosis is actually a irreversible limited hydrolysis substrate of Caspase The cascade amplification reaction process, so far, at least 14 kinds of Caspase have been found. Caspase molecules have high homology and similar structure. They are all cysteine family proteases. According to their functions, Caspase can be basically divided into Two types: one type is involved in the processing of cells, such as Pro-IL-1 and Pro-IL-1, which form active IL-1 and IL-1; the second type is involved in apoptosis, including caspase2,3,6, 7,8,9.10. The Caspase family generally has the following characteristics:

1) There is a cysteine activation site in the C-terminal homology region, and this activation site domain is QACR / QG.

2) It usually exists in the form of zymogen, with a relative molecular mass of 29000-49000 (29-49KD). After activation, its internally conserved aspartic acid residues are hydrolyzed to form two major subunits (P20) and P10 Units, and in turn form active tetramers in pairs, where each P20 / P10 heterodimer can be derived from the same precursor molecule or from two different precursor molecules.

3) The end has a small or large pro-domain.

Caspases that are involved in inducing apoptosis fall into two broad categories: inititaor and effector, which function upstream and downstream of death signaling, respectively.

Caspase Caspase activation mechanism

Caspase activation is a sequential multi-step hydrolysis process. Caspase molecules are different, but their activation process is similar. First, the N-terminal propeptide is hydrolyzed at a specific site between the N-terminal propeptide and the large subunit of the caspase precursor, and then the large and small subunits are cleaved to release the large and small subunits. The subunits constitute a heterodimer, and two dimers form an active tetramer. The removal of N-terminal propeptide is the first step of Caspase activation, and it is also necessary. However, the activation of Caspase-9 does not require the removal of N-terminal propeptide. There are basically two mechanisms for Caspase activation, namely homologous activation and heterologous activation. These two activation methods are closely related. Generally speaking, the latter is the result of the former. Caspase that undergoes homologous activation is also called initiator caspase, including caspase-8, -10, -9, which induces apoptosis. Later, the initial Caspase is recruited to a specific initial activation complex through the adaptor, forming a homodimer conformational change, resulting in enzymatic cleavage between homologous molecules and self-activation. Usually caspase-8,10,2 mediate Apoptosis leading to the death receptor pathway was recruited to the Fas and TNFR1 death receptor complexes, respectively, while Caspase-9 involved in mitochondrial pathway apoptosis was recruited to the Cyt c / d ATP / Apaf-1 Apoptosome. Homologous activation is the earliest hydrolysis and activation event of capases in the process of apoptosis. When Caspase activation is started, the death process in the cell is started, and the downstream Caspase is hydrolyzed by heterologous activation to amplify the apoptotic signal and down the death signal transfer. Heteroactivation (hetero-activation) is the classical pathway by which one type of caspase is activated and another type of caspase is activated. Caspase that is heterologously activated is also called execution caspase (executioner caspase), including Caspase-3, -6, -7. Executing Caspase is not like initiating Caspase. It cannot be recruited or bound to the initial activation complex. They must rely on initiating Caspase to activate.

Caspase Caspase effect mechanism of apoptosis

The characteristic manifestations of apoptotic cells include DNA lysis into about 200bp fragments, chromatin concentration, cell membrane activation, cell shrinkage, and finally formation of apoptotic bodies surrounded by cell membranes. These apoptotic bodies are then covered by other cells The process of phagocytosis takes about 30-60 minutes. The whole process of Caspase causing the aforementioned apoptosis-related changes is not completely clear, but it includes at least the following three mechanisms:

Apoptosis inhibitor

Normal living cells do not show DNA breaks because the nuclease is in an inactive state. This is because the nuclease is combined with the inhibitor. If the inhibitor is destroyed, the nuclease can be activated, causing fragmentation of the DNA. It is known that caspase can cleave this inhibitor to activate nuclease, so this enzyme is called caspase-activated deoxyribonuclease (CAD), and its inhibitor is called ICAD. Therefore, under normal circumstances,

Apoptosis

CAD does not show activity because CAD-ICAD exists as an inactive complex. Once ICAD is hydrolyzed by Caspase, it gives CAD nuclease activity and DNA fragmentation occurs. It is significant that CAD can synthesize and show activity only when ICAD exists, suggesting that CAD-ICAD exists in a co-transcription manner, so ICAD It is necessary to activate and inhibit CAD.

Apoptosis destroys cell structure

Caspase can directly destroy the cell structure, such as lysing the nuclear fibrous layer. The nuclear fibrous layer (Lamina) is a multimer formed by nuclear fibronectin through polymerization to form the backbone structure of the nuclear membrane. Chromatin is formed and arranged normally. When cells undergo apoptosis, lamin is used as a substrate to be cleaved by Caspase in a near-central fixed site, which causes the lamin to disintegrate and cause the shrinkage of chromatin in the cell.

Apoptosis regulatory protein function

Caspase can act on several enzymes or proteins involved in the regulation of the cytoskeleton and change the structure of the cell. These include gelsin, focal adhesion kinase (FAK), P21 activated kinase (PAK), and so on. The cleavage of these proteins leads to a decrease in their activity. For example, Caspase can cleave gelatin and generate fragments, making it impossible to regulate the cytoskeleton through actin fibers.

In addition, Caspase can inactivate or down-regulate enzymes related to DNA repair, mRNA splicing proteins, and DNA cross-linking proteins. Due to the action of DNA, the functions of these proteins are inhibited, which inhibits cell proliferation and replication and causes apoptosis.

All of this shows that Caspase "destroys" in an orderly way. They cut off the cell's connection with its surroundings, dismantle the cytoskeleton, block cell DNA replication and repair, interfere with mRNA shear, damage DNA and nuclear structure, and induce cell expression. Signals that can be engulfed by other cells and further degrade them into apoptotic bodies.

Apoptotic regulation

Apoptosis is strictly regulated. In normal cells, Caspase is in an inactive zymogen state. Once the apoptotic process begins, Caspase is activated by subsequent cascade reactions of apoptotic proteases, and irreversible apoptosis occurs. Examples of apoptosis are as follows.

Apoptosis inhibitory molecule

To date, humans have discovered a variety of apoptosis-inhibiting molecules, including P53, CrmA, IAPs, FLIPs, and apoptosis-inhibiting molecules of the Bcl-2 family.

1) P35 and CrmA are broad-spectrum apoptosis inhibitors. In vitro studies have shown that P35 forms a stable steric effect complex with target molecules in a competitive manner and inhibits Caspases activity, while P53 is at the DMQD site! G is specifically cleaved by target Caspases, and the binding of cleaved P35 to caspase is stronger. CrmA (Cytokine response modfer A) is a serum protease inhibitor that can directly inhibit the activity of a variety of proteases, but P35 has not been found in mammals. And CrmA homologous molecules.

2) FLIPs (FLICE-imhibirory proterins) can inhibit Fas / TNFR1-mediated apoptosis. It has a variety of variants, but its N-terminal functional domain (Prodomain) is exactly the same, and the C-terminal length varies. FLIPs bind to FADD and Caspase-8,10 via the DED functional domain and antagonize the interaction between them, thereby inhibiting the recruitment of Caspase8,10 to the death receptor complex and their initiation.

3) Apoptosis inhibitors (IAPs, inhibitors of Apoptosisprotien) are a group of proteins with inhibitory effects on apoptosis, first cloned from the baculovirus genome and found to inhibit the host cell death response caused by viral infection. Its characteristic is a functional region composed of about 20 amino acids, which is necessary for IAPs to inhibit apoptosis. They mainly inhibit Caspase3, -7 without binding to its zymogen. For Caspase, it can be combined with activated and In combination with zymogen, it inhibits apoptosis.

Bcl-2 Apoptotic Bcl-2 family

There are many members in this family, such as Mcl-1, NR-B, A1, Bcl-w, Bcl-x, Bax, Bak, Bad, Bim, etc. They have both anti-apoptotic and pro-apoptotic effects, respectively. . Most members have two structural homology regions, which play an important role in mediating the dimerization process between members. Dimerization between Bcl-2 members is an important form of function realization or function regulation between members. Bcl-2 physiological function is to suppress apoptosis and prolong cell life. Bcl-2 is overexpressed in some leukemias.

The subcellular localization of Bcl-2 has been clear. It can be localized on mitochondria, endoplasmic reticulum and nuclear membrane in different cell types, and exerts anti-apoptotic effects by preventing the release of mitochondrial cytochrome C. In addition, Bcl-2 has the function of protecting cells, and overexpression of Bcl-2 can cause the accumulation of nuclear glutathione (GSH), leading to changes in the redox balance in the nucleus, thereby reducing the activity of Caspase. Bax is a member of the Bcl-2 family involved in apoptosis. When apoptosis is induced, it migrates from the cytosol to the mitochondria and nuclear membrane. Some studies have found that when cytotoxic drugs induce apoptosis, the increase in nuclear membrane Bax levels is positively related to the degradation of two nuclear proteins, lamin and PARP. Cells treated with Bax oligonucleotides can only specifically block Lamin degradation and have no effect on PARP degradation. The regulatory mechanism for this effect remains unclear .

In short, the regulation of apoptosis is very complicated, and many molecules are involved. There are still many unknown mechanisms that we need to explore further.

Apoptosis medicine application

Apoptosis immunology

1) Apoptosis during thymic cell maturation: Thymic cells undergo various developmental processes and become various types of immune-active cells. In this development process, a series of positive cell selection and negative cell selection processes are involved. CD4 + T lymphocyte subtypes and CD8 + T lymphocyte subtypes are formed. At the same time, T cell clones that recognize autoantigens are selectively eliminated. The mechanism of cell clone death is mainly through programmed cell death. Therefore, the outcome of normal immune system development not only forms immune-active lymphocytes, but also produces immune tolerance to self-antigens. The formation of tolerance mechanism mainly depends on the activation of the programmed cell death mechanism of T lymphocyte clones that recognize autoantigens.

2) Activation-induced cell death (AICD) is another major type of programmed T lymphocyte death. Normal T lymphocytes are stimulated by invading antigens,

Apoptosis

T lymphocytes are activated and induce a series of immune responses. In order to prevent the immune response from being too high, or to prevent this immune response from developing indefinitely, there is AICD to control the life span of activated T cells. In fact: T lymphocyte proliferation and T lymphocyte AICD share a common signaling pathway. T lymphocytes become activated after being stimulated. After activation, T lymphocytes undergo reproductive reactions if there are growth factors, and AICD occurs if there is no or less growth factors. 3) Lymphocytes attack target cells: Immunely active cells, especially lymphokine-activated killer cells (LAK), are an important form of adoptive immunotherapy. It plays an important role in antitumor, antiviral and immune regulation. When these immune-active cells attack tumor cells and virus-infected cells, they can induce programmed cell death in target cells.

Apoptosis clinical medicine

The reason why apoptosis has become a hotspot of people's research is largely determined by the close relationship between apoptosis and clinical viruses. This relationship is not only manifested in the study of apoptosis and its mechanism, which clarifies the pathogenesis of a large class of immune diseases, and can lead to the emergence of new therapies for the disease, especially the close relationship between apoptosis and tumors and AIDS. Much attention has been paid.

1) CD4 + cell reduction caused by HIV infection is through apoptotic mechanism

HIV infection causes AIDS, and its main pathogenesis is to specifically destroy CD4 + cells after HIV infection, making CD4 + and its associated immune function defects, which easily lead to opportunistic infections and tumors, but how to specifically destroy CD4 + cells after HIV infection? ? It is generally believed that the reason for the significant decrease in the absolute number of CD4 + T lymphocytes is mainly caused by the mechanism of apoptosis. This not only clarifies the main reason for the decrease of CD4 + T cells in AIDS, but also points out an important exploration direction for the research of AIDS treatment.

2) From the perspective of apoptosis, tumorigenesis is caused by obstruction of apoptosis

It is generally believed that malignantly transformed tumor cells are caused by uncontrolled growth and excessive proliferation, and from the perspective of apoptosis, it is considered that the apoptosis mechanism of tumors is inhibited and the result of normal cell death clearance is not allowed. A series of oncogenes and proto-oncogenes are activated in tumor cells and are over-expressed. There is a very close relationship between the activation of these genes and the development of tumors. A large class of oncogenes belong to the growth factor family and a large class belong to the growth factor receptor family. Activation and expression of these genes directly stimulate the growth of tumor cells. These oncogenes and their expression products are also important for apoptosis. After the expression of many types of oncogenes in regulatory factors, it blocks the apoptotic process of tumor cells and increases the number of tumor cells. Therefore, understanding the tumorigenesis mechanism from the perspective of apoptosis is due to the apoptosis mechanism of tumor cells. Caused by reduced tumor cells. Therefore, designing a tumor treatment method based on the perspective and mechanism of apoptosis is to rebuild the apoptosis signal transmission system of tumor cells, that is, to suppress the expression of survival genes of tumor cells and activate the expression of death genes.

3) Research on apoptosis will bring real breakthroughs to autoimmune diseases

Autoimmune diseases include a large class of diseases caused by intractable immune disorders. Autoreactive T lymphocytes and B lymphocytes that produce antibodies are the main immunopathological mechanisms that cause autoimmune diseases. Under normal circumstances, the activation of immune cells It is an extremely complicated process. Under the stimulation of autoantigens, immune cells that recognize autoantigens are activated, thereby being eliminated through the mechanism of apoptosis. However, if this mechanism fails, the elimination of immune-reactive cells that recognize autoantigens will cause obstacles. Some people have observed that Fas-encoded genes are abnormal in lymphoproliferative mutant mice, and normal Fas transmembrane protein molecules cannot be translated. If Fas is abnormal, the apoptosis mechanism mediated by it is also blocked at the same time, resulting in lymphocyte proliferation. Autoimmune disease.

4) Degenerative diseases of the nervous system: Alzheimer's disease is caused by the acceleration of neuronal apoptosis. Alzheimer's disease (AD) is an irreversible degenerative neurological disease. Mutations in amyloid precursor protein (APP) presenilin-1 (PS1) and presenilin-2 (PS2) cause familial Alzheimer's disease. (FAD). Studies have shown that PS is involved in the regulation of neural cell apoptosis. Overexpression of PS1 and PS2 can enhance the sensitivity of cells to apoptotic signals. Two members of the Bcl-2 gene family, Bcl-xl and Bcl-2, are involved in the regulation of apoptosis.

Apoptotic mitochondria

Mitochondria are important organelles of eukaryotic cells, and are the main sites where animal cells produce ATP. The mitochondrial matrix tricarboxylic acid cyclic enzyme system generates NADH through substrate dehydrogenation. NADH is oxidized through the mitochondrial inner respiratory chain. At the same time, transmembrane protons are shifted to form transmembrane proton gradients and / or transmembrane potentials. ATP synthase on the inner membrane of mitochondria uses a transmembrane proton gradient energy to synthesize ATP. The synthesized ATP is exchanged into the cytoplasm through the mitochondrial inner membrane ADP / ATP carrier into the cytoplasm and participates in various energy demand processes of the cell.

In 1951, Glucksmann, honorary professor at the University of Paris VIII, proposed cell death in normal vertebrate development. In 1966, Saunders proposed cell death during morphogenesis. In 1972, Kerr proposed apoptosis, indicating that this is a basic biological phenomenon that has a broad role in tissue dynamics. In 1974, Lockshin proposed programmed cell death. Horvitz, a professor at the Massachusetts Institute of Technology in the United States, studied the development of nematodes and found that the position, division and fate of each cell of the nematode are precisely determined by genetically determined procedures. When forming adult bodies, 1090 cells were born and 131 cells died. In 1993, Yuan Junying, a tenured professor in the Department of Cell Biology at Harvard Medical School, discovered that the product of ced-3, the death gene of nematodes, is structurally and functionally homologous to mammalian interleukin-1 converting enzyme. Since then, more than a dozen related genes belonging to the same family have been found in the mammalian genome. Collectively called caspases. In 1994, Hengartner, Institute of Molecular Life Sciences, University of Zurich, Switzerland, found that the product of the survival gene ced-9 of the nematode is similar to that of the mammalian proto-oncogene bcl-2.

Apoptosis is characterized by the cell tending to die in the nearly normal plasma membrane due to the action of degrading enzymes, mainly hydrolases (proteases and nucleases). This is different from the early damage of the plasma membrane during necrosis. During the apoptotic process, the plasma membrane lipid bilayer loses bilateral asymmetry, and phosphatidylserine is exposed on the cell surface, which leads to its engulfing.

Close relationship between the dissipation of apoptosis mitochondrial transmembrane potential and apoptosis

There have been successive reports that the mitochondrial transmembrane potential is dissipated earlier than nuclease activation and also before phosphoryl acylserine is exposed to the cell surface. Once the mitochondrial transmembrane potential is dissipated, cells will enter an irreversible apoptotic process. Mitochondrial dissociated respiratory chains produce a large amount of reactive oxygen species, which oxidizes cardiolipin on the inner membrane of mitochondria. Experiments have shown that mClCCP can cause lymphocyte apoptosis. And if you can stabilize the mitochondrial transmembrane potential, you can prevent apoptosis.

Apoptotic permeability transition

Dissipation of the mitochondrial transmembrane potential during apoptosis is mainly due to the permeability transition of the inner membrane of the mitochondria, which is due to the generation of a dynamic communication composed of multiple proteins located at the inner and outer membrane contact sites of the mitochondria. Permeability transition channel (PT channel) (Figure 1). The PT channel is composed of proteins in various parts of the mitochondria and proteins in the cytoplasm. This includes cytosol proteins: hexokinase, mitochondrial outer membrane proteins: peripheral benzodiazepine receptors and voltage-dependent anion channels, mitochondrial membrane gap proteins: creatine kinase, mitochondrial membrane proteins: ADP-ATP carrier , Mitochondrial matrix proteins: cyclophilin D, etc. Any substance that can specifically act on mitochondria to induce PT channel formation, such as the ligand protoporphyrin IX of the benzodiazine receptor, can cause apoptosis.

PT The nature of apoptotic PT channels

Through some laboratory research, the following points are worth pointing out: The transition of mitochondrial inner membrane permeability is not only a necessary condition for apoptosis, but also a sufficient condition for it. The opening of the PT channel leads to fatal changes in many functions of the mitochondria and thus initiates the death pathway. PT channel as a sensor of many physiological effects (divalent cations, ATP, ADP, NAD, m, pH, thiol and peptides), integrates electrophysiology, redox and cell metabolism status information. The component of PT channel ADP-ATP vector is an important molecule for energy metabolism. Since ADP-ATP vector is encoded by several members of a gene family, its expression has strict tissue specificity. Therefore, the regulation of PT channels in different cells may be slightly different. The effect of PT channel has the effect of self-amplification. PT induces m dissipation, while mClCCP depolarizing m in turn causes PT. Some PT results such as m are dissipated, and the generation of active oxygen itself can also cause PT. This shows that PT will have positive feedback, and thus have a self-destructive effect on apoptosis. Conversely, if the dissipation of m can be prevented, the redox imbalance, the exposure of phosphorylserine and the activation of proteases and nucleases can be avoided.

PT Apoptotic PT channel switch

PT tunnels have two conformations, open and closed. The opening of the PT channel leads to apoptosis. The closing of the PT channel can prevent apoptosis. The PT channel is closed when it is combined with cyclosporin A or SH or bongkrek acid. Mitochondria release apoptosis-inducing factor (AIF) when the PT channel is opened. AIF may be a proteolytic enzyme, located in the intermitochondrial space, and it can be inhibited by protease inhibitors such as N-benzyloxycarbonyl-valyl -Ala-Asp-fluoromethylketone). In addition, cytochrome C released from mitochondria is also an apoptosis-inducing factor. Although atractylodesin and fulvic acid are both inhibitors of the ADP-ATP carrier, they have different effects on the PT channel. Atractyloside promotes the opening of PT channels. This may be related to the different binding sites of the two inhibitors and the ADP-ATP vector. Atractylglycoside can only bind to the cytosol side of the ADP-ATP carrier, and fulvic acid can bind to the cytosol and matrix sides of the ADP-ATP carrier.

Apoptotic mitochondria

If the purified normal mitochondria are incubated with the purified cell nucleus, it will not cause changes in the cell nucleus. However, if the mitochondria that induce PT channels are incubated with the purified nuclei, the nuclei will begin to change. Regardless of whether it is the bcl-2 family that inhibits death or the Bax family that promotes cell death, mitochondria are used as target organelles. The C-terminal hydrophobic peptide of the bcl-2 protein can be inserted into the outer mitochondrial membrane. In fact, a considerable amount of bcl-2 is located at the contact site of the outer and inner membranes of the mitochondria. (3) High expression of bcl-2 can prevent m dissipation, which leads to insensitivity to atractin, protoporphyrin IX and mClCCP and inhibition of AIF release; conversely, high expression of Bax causes m dissipation.

In summary, apoptosis is closely related to the structure and function of mitochondria. If a large number of PT channels are formed in the mitochondria and the ATP concentration of the cells decreases quickly, the cells will die before the apoptotic proteases are activated. And if the induced generation of PT channels is a relatively mild and continuous state, a specific protease is activated before the ATP concentration of the cell decreases; on the other hand, the superoxide anion produced by the dissipation of m results in cell death. Apoptosis is a double-edged sword. On the one hand, it is a normal process of body development; on the other hand, if the apoptosis is too fast, it will lead to chronic degenerative diseases; if the cells are not apoptotic, it may cause canceration or insensitivity to chemotherapy. Further research on the role of mitochondria in apoptosis will help to understand the mechanism of apoptosis and prevent and cure diseases.

Apoptosis detection

Early detection of apoptosis

1) Detection of PS (phosphatidylserine) on the outer membrane of cells:

Apoptosis

The transfer of PS from the inside to the outside of the cell membrane occurs shortly after the cell is induced by apoptosis and may serve as a recognition marker for the immune system. AnnexinV, a calcium-dependent phospholipid-binding protein, specifically binds to PS exposed outside the membrane, and can be detected by a simple color or luminescence system. Since this is a live cell test for early apoptosis (both suspended cells and adherent cells), it can be combined with DNA dyes or other advanced detection methods to mark the stage of development of apoptosis.

American well-known biological reagent company CLONTECH and Invitrogen have developed a variety of labeled Annexin V products, which is simple and fast, and can be detected in 10 minutes. Among them, fluorescently labeled Annexin V-EGFP (Enhanced Green Fluorescent Protein) and Annexin V-FITC have high sensitivity and can be used as the basis for screening apoptotic cells by FACS (flow cytometry) method. Due to the fusion protein Annexin V-EGFP, the binding ratio of EGFP to PS is 1: 1, which can also be quantitatively detected. In addition, biotin-conjugated Annexin V is also available, which can be detected by common enzyme-linked color reaction. In addition, MACS company coated magnetic beads with Annexin V, and apoptotic cells can be screened by magnetic sorting.

2) Detection of changes in intracellular redox status:

This reflects a relatively new trend in the study of apoptosis, which studies what redox environment causes downstream events. CLONTECH's ApoAlertTM Glutathione Detection Kit detects the change of redox status in early apoptotic cells by detecting the reduction of glutathione in the cytoplasm of apoptotic cells in vitro by the fluorescent dye monochlorobimane (MCB). Under normal conditions, glutathione (GSH) acts as an important redox buffer for cells. Toxic oxides in cells are periodically removed by reduction by GSH, and oxidized GSH can be quickly reduced by GSH reductase. This reaction is particularly important in mitochondria, where many oxidative damage of by-products from respiration will be removed. In Jurcat and some other cell types, there is an ATP-dependent GSH transfer system in the cell membrane that can be activated by an apoptotic signal. When the elimination of GSH in cells is very active, the cytosol changes from a reducing environment to an oxidizing environment, which may lead to a decrease in the mitochondrial membrane potential of the early apoptotic cells, thereby causing cytochrome C (an important component in the tricarboxylic acid cycle ) Transfer from the mitochondria to the cytosol, initiating a cascade of apoptotic effector caspase.

Because GSH is closely related to redox and mitochondrial function, this test is very useful for studying the onset of apoptosis, and it can also be used for the treatment of diseases such as heart disease and stroke. However, some cells, such as HeLa and 3T3 cells, have no obvious changes in GSH levels during apoptosis and cannot be detected by this method.

3) Localization detection of cytochrome C

As a signaling substance, cytochrome C plays an important role in apoptosis. Under normal circumstances, it exists in the cavity between the inner and outer membranes of the mitochondria, and the apoptotic signal stimulates it to release from the mitochondria to the cellular fluid. In combination with Apaf-1 (apoptoticprotease activating factor-1), the caspase cascade is initiated: The cytochrome C / Apaf-1 complex activates caspase-9, which in turn activates caspase-3 and other downstream caspase. Cytochrome C oxidase subunit (COX4) is a membrane protein localized on the inner membrane of mitochondria. When apoptosis occurs, it remains in the mitochondria, so it is a very useful part of mitochondrial enrichment. symbols of.

The ApoAlertTM Cell Fractionation Kit does not require ultracentrifugation, and can quickly and efficiently isolate highly enriched mitochondrial fractions from apoptotic and non-apoptotic cells. The location of cytochrome C and COX4 is further marked by Western blot with cytochrome C and COX4 antibodies To determine the occurrence of apoptosis.

4) Detection of mitochondrial membrane potential changes:

Early in the apoptotic studies, there were no significant changes in mitochondria from morphological observations. With the further study of the mechanism of apoptosis, it was found that mitochondrial apoptosis is also an important part of apoptosis, and many physiological and biochemical changes have occurred. For example, mitochondrial transmembrane potential changes after being induced by apoptosis, leading to changes in membrane permeability. MitoSensorTM, a cationic stain, is very sensitive to this change and exhibits different fluorescent stains. In normal cells, it forms aggregates in the mitochondria, emitting intense red fluorescence. In apoptotic cells, due to changes in the mitochondrial transmembrane potential, it exists in the cell fluid as a monomer and emits green fluorescence. These two different fluorescence signals can be clearly resolved using a fluorescence microscope or a flow cytometer. CLONTECH's ApoAlert Mitochondrial Membrane Sensor Kit uses this principle to detect changes in mitochondrial membrane potential. However, this method cannot distinguish between changes in mitochondrial membrane potential caused by apoptosis or other reasons.

Advanced apoptosis detection

In the late stage of apoptosis, endonucleases (substrates of some caspases) cut nuclear DNA between nucleosomes, producing a large number of 180-200 bp DNA fragments. There are two methods to detect this phenomenon:

1) TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)

The labeled dNTP (mostly dUTP) is directly (through digoxin) or directly connected to the 3'-OH end of the DNA fragment by DNA terminal transferase, and the results are quantitatively analyzed by enzyme-linked color development or fluorescence detection. American Intergen company provides a variety of labeling methods, direct fluorescent labeling, digoxin-mediated fluorescent labeling or peroxidase-linked color development, can be used for cell suspensions, formalin-fixed or paraffin-treated tissues, cell cultures, etc. Detection of multiple samples. Among them, there are few direct labeling steps and easy operation. The indirect marker has the function of signal amplification and high detection sensitivity.

2) LM-PCR Ladder (Ligation-mediated PCR)

When the proportion of apoptotic cells is small and the amount of test samples is small (such as biopsy tissue sections), changes in nuclear DNA may not be observed by direct agarose electrophoresis. CLONTECH's ApoAlert LM-PCR Ladder Assay Kit uses LM-PCR (ligation-mediated PCR) to connect specific adapters to specifically amplify gradient fragments of nucleosomes to sensitively detect apoptosis. Fragments of nucleosomes. In addition, LM-PCR detection is semi-quantitative, so different samples with the same degree of apoptosis can be compared.

The above two methods are aimed at the characteristic of nuclear DNA breakage at the late stage of apoptosis, but other damages to the cell (such as mechanical damage, ultraviolet rays, etc.) will also cause this phenomenon, so its detection of apoptosis will be affected by other reasons. interference.

3) Telemerase Detection

This is a relatively early method, and it is used more often. Telomerase is a nucleoprotein composed of RNA and protein. It can reverse-transcribe the telomere repeat sequence with its own RNA as a template, so that the cell can obtain "immortalization". Normal somatic cells do not have telomerase activity, and each time they divide, the telomeres of the chromosomes are shortened, which may serve as a clock for mitosis, indicating a signal of cell age, replicating senescence, or apoptosis. Studies have found that more than 90% of cancer cells or apoptotic cells have telomerase activity. Invitrogen's TRAP-eze Telemerase Detection Kit was first introduced in 1996. It provides specific oligonucleotide substrates, primers that pair with the substrate and telomere repeats, respectively. If the test sample contains telomerase activity, a different number of 6 base (GGTTAG) telomere repeats can be connected to the substrate. By PCR reaction, the product electrophoresis detection can observe a difference of six bases DNA Ladder phenomenon (see Figure 4). In addition, Intergen also provides kits for detection by enzyme-linked immunoassay (ELISA).

Similarly, this test method is not specific to apoptosis, and the test results do not reflect the occurrence of apoptosis.

mRNA Apoptotic mRNA detection

Researchers have found many genes that express abnormally during apoptosis, and detecting the expression levels of these specific genes has also become a common method for detecting apoptosis. It is reported that Fas protein can induce target cells such as cytotoxic T cells in cancer cells after binding to receptors. Bcl-2 and bcl-X (long) are regulators of anti-apoptosis (bcl-2 and bcl-X), and their expression level ratio determines whether the cell is apoptotic or viable. They are usually detected by Northern hybridization and RT-PCR gel removal. With the development of fluorescent quantitative PCR technology, quantitative PCR technology is no doubt faster and more accurate than the former. Invitrogen's Amplifluor Apoptosis Gene Systems uses this new technology principle to detect apoptosis by detecting the mRNA expression levels of fas, bax-alpha and bcl-X (long) genes.

Apoptosis Morphology

Morphological detection of apoptosis

According to the inherent morphological characteristics of apoptotic cells, many different morphological detection methods for apoptotic cells have been designed.

1. Light and inverted microscopes

1. Unstained cells: The apoptotic cells become smaller and deformed, the cell membrane is intact but foaming occurs, and apoptotic bodies can be seen in the later stages of apoptosis.

Adherent cells shrink, round, and fall off.

2. Stained cells: Giemsa staining and Wright staining are commonly used. Chromatin concentration and marginalization of apoptotic cells, nuclear membrane lysis, chromatin segmentation

Typical apoptotic morphology such as lumps and apoptotic bodies.

2. Fluorescence microscope and confocal laser scanning microscope

The morphological changes of nuclear chromatin are generally used as indicators to evaluate the progress of apoptosis.

Commonly used DNA-specific dyes are: HO 33342 (Hoechst 33342), HO 33258 (Hoechst 33258), DAPI. The binding of the three dyes to DNA is non-embedded, mainly in the AT base region of DNA. UV light emits bright blue fluorescence when excited.

Hoechst is a reactive dye that specifically binds to DNA. The storage solution is made up to a concentration of 1 mg / ml with distilled water. When used, it is diluted with PBS to a final concentration of 2 to 5 mg / ml.

DAPI is semi-permeable and used for staining of conventional fixed cells. The storage solution is made up to a concentration of 1 mg / ml with distilled water, and the final concentration is generally 0.5 to 1 mg / ml.

Result evaluation: The morphological changes of nuclear chromatin during apoptosis are divided into three phases: phase nucleus is rippled or creased, and some chromatin appears concentrated; phase a Chromatin is highly condensed and marginalized; the nucleus of phase IIb lyses into fragments, producing apoptotic bodies.

3 Transmission electron microscope observation

Judgment of results: Apoptotic cells became smaller in volume and cytoplasm was concentrated. The chromatin in the nucleus of the pro-apoptosis nuclei stage is highly coiled, and many vacuole structures called cavitations appear; the chromatin in the nucleus of the stage IIa is highly condensed and marginalized; the late stage of apoptosis The nucleus is lysed into fragments, producing apoptotic bodies.

Apoptosis detection

Apoptosis plays an important role in embryonic development, hematopoiesis, the maturation of the immune system, and the maintenance of cell constant and growth balance in normal tissues and organs, and even the aging of the body. Therefore, research on apoptosis has been widely carried out in various fields such as clinical and basic, and the detection method of apoptotic cells is very important. Flow cytometry (FCM) integrates fluid ejection technology, laser optics technology, electronic technology and computer technology, which has incomparable advantages over other methods, both qualitative and quantitative, and simple, Fast and sensitive, it can be used for multi-parameter and live cell analysis. The research in APO has been widely used, opening up new ways.

1 Light scattering method

In the FCM system, when the tested cells pass through the measuring area of the instrument in the liquid flow, after the laser is irradiated, the cells scatter light in all directions of the solid angle of 360 ° in space. The intensity of the forward scattered light (FSC) is related to the cell size. The intensity of side-scattered light (SSC) is related to the refractive index of the plasma membrane and the interior of the cell. During apoptosis, the cells shrink, the volume becomes smaller, the nuclear fragmentation is formed, and the intracellular particles tend to increase. Therefore, the apoptotic cells have reduced FSC and increased SSC. Cell necrosis Due to the swelling of the cell body, the nucleus also fragmented and decomposed, so FSC and SCC increased. Normal cells have high FSC and low SSC. The main advantage of detecting apoptotic cells based on light scattering characteristics is that light scattering characteristics can be combined with cell surface immunofluorescence analysis to distinguish and identify lymphocyte subtypes that undergo selective apoptosis after these special treatments. Cell classification. It is worth noting that the reliability of judging apoptotic cells according to FSC and SSC is greatly affected by the morphological uniformity of the tested cells and the ratio of nuclear cytoplasm.Therefore, in some lymphocyte apoptosis, the light scattering characteristics are used to detect The reliability of death is better but its reliability is lower in tumor cell apoptosis.

2 Determination of cell DNA content

During apoptosis, endonucleases are activated, leading to DNA fragmentation. This is a characteristic manifestation of apoptosis and also lays the foundation for FCM to identify apoptotic cells. Among the methods for detecting apoptotic DNA breaks, the most common and simplest method is the analysis of cellular DNA content. When cells were treated with ethanol and TrtionX-100, loopholes appeared in the cell membrane, and small fragments of DNA were released from the cells, making their DNA content lower than that of diploid cells in normal cells. After analysis with propidium iodide (PI) staining, a diploid C0 / G1 peak can appear before the peak, that is, the apoptotic peak (APO peak). Apoptosis can be measured based on the APO peak Cell percentage, this method is simple and easy, can be used to quantitatively detect apoptotic specimens, and can also analyze the cell cycle position of cells. In addition, FCM method can be used to identify G0 phase cells through the combined detection of DNA and RNA. Therefore, the relationship between apoptosis and G1 or G0 thin chest can be analyzed. The degree of DNA degradation depends on the stage of apoptosis, the type of cell, and the characteristics of the apoptosis-inducing factors. Changes in DNA escape during staining also affect FCM results. According to research, adding a high concentration of phosphate-citrate buffer to the rinse solution can increase the escape amount of degraded DNA, thereby improving the ability to distinguish apoptotic cells from normal cells.

The limitation of DNA content measurement in detecting apoptosis is that its specificity and sensitivity are not high. The specificity is not high because the APO peak represents a group of cell populations, including apoptotic cells, mechanically damaged cells, cells with low DNA content, or cells with different chromosomal structures.In the above cases, the amount of DNA and fluorescent dye binding is small . In addition, when non-fixed cells are lysed in a hypotonic solution, a large number of nuclear fragments can appear. At this time, the number of APO peak cells only represents the number of nuclear fragments, not the number of apoptotic cells. The reason for the poor sensitivity is that only DNA breakpoints appear in the early stage of apoptosis, but a large number of DNA fragments have not yet been lost, so this method cannot detect early apoptotic cells and apoptotic cells that occur in S or G2 / M phases. Because its actual content is not less than the DNA contained in diploid cells, the analysis of apoptotic cells should be combined with other morphological or biochemical methods in order to more accurately analyze the apoptotic status of cells.

3 Y Acridine Orange (AO)

AO stains double-stranded DNA and denatured DNA in cells or nuclei with different colors of fluorescence. When AO is inserted into double-stranded DNA, it emits green fluorescence; AO can also interact with single-stranded or single-stranded DNA generated by denaturation, and then emit red fluorescence. Therefore, the different fluorescence can be detected by FCM to determine the apoptosis. occur. After the measurement is standardized, the amount of green and red fluorescence intensity is proportional to the total DNA content, and the ratio of red fluorescence to overall cell (red plus green) fluorescence indicates the proportion of denatured DNA in the cell, so this method can be used to evaluate DNA Sensitivity to in situ degeneration. Sometimes, the DNA degradation of apoptotic cells is not obvious, and it is difficult to detect apoptosis changes by methods that rely on DNA degradation to detect apoptosis, such as determination of cell DNA content and DNA end markers. The principle of AO method for detecting apoptosis does not depend on the generation of DNA fragments, so its main advantage is that it can be applied to situations where oligonucleosome fragments are not in equilibrium with apoptosis, but the AO staining method cannot effectively distinguish between mitotic cells and withered cells. Dead cells.

4 Rhodamine (Rh123) staining

In the state of cell life, the role of sodium-potassium pump and calcium pump on the cell membrane maintains different ion concentration gradients inside and outside the cell membrane, including Na +, K +, Cl-, Ca2 +, etc., forming cell membrane potential. FCM can detect the distribution of lipophilic ionic fluorescent dyes inside and outside the cell membrane to measure the level of membrane potential to evaluate cell viability. Rh123 is a lipophilic cationic fluorescent dye, which has permeability to cell membranes, and is particularly sensitive to mitochondrial membranes. When the cells are alive, rhodamine 123 accumulates in the mitochondria through the cell membrane and emits green fluorescence. During apoptosis, the mitochondrial membrane's transport capacity decreases and its electronegativity decreases, so the ability of cells to accumulate Rh123 is also lost, and the fluorescence intensity is reduced. Based on this, the changes in apoptosis of cells are detected.,,,,,Rh123

5 In situ end-labeling technology

During apoptosis, DNA breaks earlier than morphological changes and DNA content decreases.In situ end-labeling (ISEL) is the exogenous nucleotide that penetrates into apoptotic cells under the catalysis of enzymes and DNA. The combination of single-strand or double-strand breaks due to the activation of endogenous nucleases is more sensitive than the aforementioned aspects. There are usually two methods: DNA polymerase I or klenow large fragment-mediated unit gap translation (INST); terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).

INST uses DNA polymerase to integrate nucleotides to the 3 'end of broken DNA in apoptotic cells, and hydrolyzes the 5' end to repair DNA. If labeled nucleotides are used, they can show broken DNA. cell. In 1993, Gorczyca et al. Proposed a terminal deoxyribonucleic acid transferase (TdT) labeling method to detect DNA breaks in apoptotic cells. This method has been widely used. Due to the activation of endogenous endonucleases, the cell's own chromatin or DNA is cleaved and 3'2 hydroxyl termini are generated as many as DNA breakpoints. TdT can label biotinylated dUTP to the 3'2 hydroxyl terminus Through the avidin 2FITC system, specific breakout sites of DNA are identified to identify apoptotic cells. TdT end labeling is a more specific method for identifying apoptotic cells. There are few apoptotic cells in brain tissue, so genomic DNA fragments require more sensitive detection techniques. Combining TUNEL with FCM can increase the sensitivity of detecting DNA fragments in apoptotic cells. Cells treated with apoptosis-inducing factors for a certain period of time showed more in situ end-labeled apoptosis than Hoechst33342 staining, suggesting that TUNEL can detect nuclei that have not yet exhibited obvious morphological characteristics of apoptosis but have undergone DNA lysis, thereby Increased detection sensitivity. Comparative studies show that the sensitivity of TUNEL is much higher than that of ISNT, especially in the early stage of APO, the positive rate of TUNEL method is high, which may be the reason that most of the DNA is double-stranded and broken at the same time when APO occurs, and single-strands are rare. The latter is dependent on DNA polymerase-mediated repair reactions, so the positive rate of ISNT is relatively low. TUNEL can also be combined with simultaneous analysis of cells to understand the relationship between apoptotic cell DNA breaks and cell cycle distribution. Recently, TUNEL has become one of the most commonly used methods for identifying and quantifying apoptotic cells. However, because the process of labeling broken DNA is complicated and involves many factors, the negative result of end labeling does not necessarily represent the integrity of the DNA strand. Methodological issues, such as the loss of TdT enzyme activity and many other factors, should be ruled out. Therefore, the identification of apoptotic cells by TdT terminal labeling method must be set up with both positive and negative control groups in order to obtain reliable results.

6 Annexin V / PI method

In 1992, Fadok reported that phosphatidylserin (PS), which is located on the inner side of the cell membrane, migrated to the outside of the cell in the early stage of APO. This phenomenon occurred before nuclear chromatin degeneration and nuclear volume shrinkage. AnnexinV is a vascular protein with strong anticoagulant properties. It has a high affinity for phospholipids, and especially has a strong binding force with negatively charged phospholipids such as PS. Using its characteristics can detect cell apoptosis. However, the necrotic cell PS is also exposed to the appearance to make Annexin V bind positively. Therefore, the parameter of Annexin V cannot be used to distinguish between necrosis or apoptosis. It is necessary to use PI as a parameter to distinguish necrosis from the biliary. FCM analyzes apoptotic and necrotic cells through Annexin V-FITC markers exposed to PS and PI on the cell membrane combined with PI to enter the damaged cell membrane to mark and degrade DNA. There are 4 subpopulations in the test including mechanically injured cells (Annexin-/ P1 +), normal cells (An2nexin-/ PI-), apoptotic cells (Annexin + / PI-), and secondary necrotic cells (Annexin + / PI +) are distinguished. Boersma et al. Used Ampexin V2FITE staining to detect apoptosis in Chinese hamster cells treated with cytotoxic drugs. FCM detection revealed two cell subpopulations with different fluorescence signals. Further morphology confirmed that the weakly fluorescent cell subgroup represents early apoptotic cells, and the strong fluorescent subgroup represents late apoptotic cells, which shows that it is a more reliable method for detecting and quantifying apoptotic cells. During cell apoptosis, PS exposure on the membrane occurs earlier than DNA breakage, so this method is more sensitive to detect early apoptosis, and this method does not require fixed cells, which avoids excessive cell debris caused by PI method and TUNEL method due to fixation. The appearing DNA fragments are lost, so it is more time-saving and the results are more reliable. It is currently the most ideal method for quantitative apoptosis detection.

7 other

7.1 ssDNA monoclonal antibody methodThe use of anti-single-stranded DNA (ssDNA) monoclonal antibodies for the detection of apoptosis is an accidental discovery, because in the application of ssDNA monoclonal antibodies (fluorescence method) to detect cytotoxic drugs-induced DNA damage, observation The apoptotic leukemia cells (MOL T24) had strong fluorescence, and after proper improvement, it was proved that ssDNA monoclonal antibodies can specifically recognize apoptotic cells. Compared with the TUNEL method, ssDNA has stronger sensitivity. Apoptotic cells detected by TUNEL method may be just a subtype of apoptotic cells detected by monoclonal antibody method. The ssDNA method is generally used to detect APO by immunofluorescence. But it can also be combined with FCM. The monoclonal antibody method is simple to use, low in cost, and widely used. ssDNA monoclonal antibodies can distinguish between necrosis and apoptosis, and can even detect pre-apoptosis, post-apoptosis necrosis and some special forms of apoptosis (such as non-fragmented cell apoptosis). Therefore, the ssDNA monoclonal antibody method is expected to become a new and sensitive method for detecting apoptosis.

7.2 Analysis of apoptosis-related proteins found that there are many genes involved in the regulation of apoptosis, and these gene products can participate in promoting or inhibiting the occurrence and development of APO. . So far, a large number of protein products of apoptosis-regulating genes have been analyzed, such as P53 protein, caspases, C2myc, Fas antigen, TNF, bcl22 family proteins, cyclin, ras, etc. FCM stains with a variety of fluorescently labeled regulatory monoclonal antibodies, collects fluorescent signals of different wavelengths, detects the number of fluorescent molecules on the surface of the cell membrane or in the cell, and can understand the changes of each cell. .

8 Outlook

In recent years, with the continuous development of FCM technology and the gradual deepening of APO research, FCM has become increasingly widespread in the study of apoptosis. Using FCM to quantitatively detect apoptotic cells is simple, fast, and objective, and can perform multi-parameter detection.Therefore, APO and its related oncogene expression, cell cycle distribution, and many other factors can be analyzed at the same time, which can provide a deeper understanding of the disease. Regulatory mechanism. Although there are many methods for the study of apoptosis using FCM, the method for detecting apoptotic cells by FCM is generally based on some aspects of morphology, biochemistry, etc. during the apoptosis process, so it is difficult to understand the various changes that occur during the apoptosis The interrelationship also makes this type of method lack specificity. Therefore, combined application of multiple FCM detection methods for different characteristics can more effectively identify apoptotic cells. At the same time, FCM research results need to be combined with morphological observation or biochemical methods to understand the biological characteristics of apoptotic cells more deeply. With the development of biotechnology and people's deep understanding of the nature of APO, I believe that in the near future, there will be more specific and sensitive methods coming out, which will help breakthrough progress in apoptosis.

Significance of apoptosis

Apoptosis and cell proliferation are basic phenomena of life, and are the basic measures to maintain the dynamic balance of the number of cells in the body. The elimination of excess and completed cells through apoptosis during embryonic development ensures the normal development of the embryo; the elimination of senescent and diseased cells through apoptosis during adulthood ensures the health of the body. Like cell proliferation, apoptosis is also a precise process regulated by genes.