What Is Fetal Blood?

Generally refers to the blood in human embryos, which contain endothelial progenitor cells.

Fetal blood

Right!

Generally refers to the blood in human embryos, which contain endothelial progenitor cells.

Chinese name

Fetal blood

Foreign name

endothelialprogenitorcells

Category

Endothelial progenitor cells

Short name

EPCs

To study the differentiation of human umbilical cord blood mononuclear cells into vascular endothelial progenitor cells (EPCS) induced by VEGF and bFGF

Mononuclear cells were obtained from fresh umbilical cord blood by Ficoll density gradient centrifugation, cultured in M199 medium supplemented with VEGF and bFGF, cultured for 7 days, and immunohistochemistry (SABC method) and immunofluorescence were used to detect cell surface antigen expression.

Induction of human umbilical cord blood mononuclear cells (MNCS) by VEGF, bFGF in vitro

Human umbilical cord blood mononuclear cells can be induced to differentiate into endothelial progenitor cells under certain culture conditions.

Endothelial progenitor cells (endothelialprogenitorcells, endothelialprogenitorcells EPCs) are a type of endothelial progenitor cells that can proliferate and differentiate into vascular endothelial cells, but have not yet expressed the mature vascular endothelial cell phenotype and have not formed vascular precursor cells [1]. Studies have found that EPC is not only involved in human Embryo angiogenesis also participates in the process of postnatal angiogenesis and repair after endothelial injury. Therefore, EPCs have broad clinical application prospects in coronary heart disease, tumors, and wound healing. This year, EPCs have been used in basic research and cardiovascular diseases. Research in the fields of limb ischemic diseases is very active. The foreign research on EPCs is in-depth, and the research on EPCs in China is still in its infancy. We isolate and culture human cord blood, expand it in vitro, and observe the in vitro differentiation of EPCs. Process to lay the foundation for further research of EPCs.

1 Materials and methods 1.1 Material sucrose diatrizoglucamine with a specific gravity of 1.077 ± 0.001 (trade name is lymphocyte separation solution). D Hanks solution (Ca2 + -free, Mg2 +). VEGF and bFGF are from Sigma. Fetal bovine serum (Hangzhou Sijiqing Company). Mouse anti-human CD34 monoclonal antibody, rabbit anti-human KDR monoclonal antibody, FITC-labeled goat anti-mouse, goat anti-rabbit secondary antibody, mouse anti-human CD133 monoclonal antibody (SantaCruz). M199 culture solution, immunohistochemistry Staining kit (ready-to-use SABC) (Wuhan Bode Bioengineering Co.). SantaCruzBio fluorescence microscope; OlympusCK40 inverted microscope; Low-temperature high-speed centrifuge Biofuge22R.
1.2 Method 1.2.1 Collection and separation of EPCs Human umbilical cord blood was collected from term healthy maternity women, and each blood collection was about 40 ~ 60mL. Compound sodium citrate injection (acda) was added to anticoagulant at 1:10. Then 1: 1 Diluted with PBS. EPCs were separated by Ficoll density gradient centrifugation. After centrifugation at 2000 r / min, the tube was divided into three layers after centrifugation. The white cloud layer with a single mononuclear cell at the upper and middle interface was placed in another centrifugation zone and centrifuged. In the tube, the cells were washed by centrifugation with Hanks solution twice. After the last centrifugation, the supernatant was discarded, and the cells were resuspended in M199 medium (containing 100 mL / L fetal calf serum, cytokines growth factor VEGF and bFGF). Take a drop of the cell suspension with A drop of 2g / L trypan blue staining solution was mixed to calculate the concentration of mononuclear cells and the detection of cell viability.
1.2.2 Culture of EPCs Mononuclear cells were seeded at a density of 5 × 108 / L into a 10g / L gelatin-plated culture flask, placed in a cell incubator at 37 ° C, 50mL / LCO2, and 100% humidity, and discarded after 48h Remove adherent cells, resuspend the suspended cells with M199 medium, and inoculate them at a density of 2 × 108 / L into a six-well plate with coverslips. Change the solution every 3 days and wash off the unattached with Hanks solution Climbing cells were collected on day 7 of the culture for identification.
1.2.3 Immunofluorescence detection: Climbing cells on the seventh day of culture were fixed at 40 g / L paraformaldehyde for 20 min, and then washed 3 times with PBS for 5 min each. After drying, 100 L of FITC direct-labeled mouse anti-human CD133 monoclonal antibody was added. (1: 50-fold dilution), put in a wet box and incubate at 37 ° C for 1h, and replace the CD133 monoclonal antibody with PBS as a negative control. Then wash twice with PBS, wash once with distilled water, seal, and observe under a fluorescence microscope.
1.2.4 Immunohistochemical detection Climbing cells were fixed with 40g / L paraformaldehyde for 20min on the 7th day of culture, endogenous peroxidase was inactivated with 30ml / LH2O2, nonspecific binding was blocked with 30ml / LBSA, antibodies Rabbit anti-human KDR and mouse anti-human CD were kept at 34.4 overnight, developed with SABC method, counterstained with hematoxylin, dehydrated, mounted on slides, and observed the results under an inverted microscope.
2 Results The mononuclear cells obtained from fresh umbilical cord blood were round, and the cell viability was 99%. The isolated umbilical cord blood mononuclear cells adhered after 48 h of culture and grew at 4 to 5 days. The fastest, a large number of spindle cells appeared about 5 days of culture. After 5 days of mononuclear cell culture, a large number of cell clusters were formed under the microscope. About 6 days of culture, long spindle cells were seen to grow radially with the cell cluster as the center. Scattered long spindle cells can also be seen. When cultured for 7 days, it can be seen that multiple long spindle cells are connected end-to-end to form a strip-like arrangement, and sometimes two rows of cells are arranged in a tube-like structure in parallel (Figure 1). For KDR, Immunofluorescence and immunohistochemical detection of surface markers such as CD133 and CD34. Immunohistochemistry showed CD34, KDR positive staining. Immunofluorescence showed CD133 positive staining of cells, yellow-green fluorescence was seen in the cell membrane and cytoplasm under a fluorescence microscope (Figure 2) ).
A: cell cluster (5d); B: long spindle-shaped cells forming band-like (7d) × 200.
Figure 1 Peripheral mononuclear cell culture Figure 2 Immunofluorescence detection of CD133 positive staining (7d) × 400 (omitted)
3 Discussion Ashara et al. [3] isolated and cultured EPCs from peripheral blood in 1997, and proved that EPCs are also involved in post-natal angiogenesis. This discovery provides a new treatment plan for ischemic diseases, which can be mobilized , In vitro expansion and transplantation of EPCs to promote the formation of new blood vessels. This experiment intends to establish a relatively simple and reliable method to obtain EPCs in preparation for the clinical application of EPCs.
At present, there are many methods for the isolation of EPCs in vitro, mainly including density gradient centrifugation and immunomagnetic bead sorting. Some scholars have combined the two methods, and then centrifuged and then separated by immunomagnetic beads. Density gradient centrifugation The operation is simple and convenient, but there is impure separation, which can only isolate the whole monocytes (including EPCs) in the blood, including some platelets. The immunomagnetic bead sorting method generally uses CD34 antibody in the early stage. The more scholars use the CD133 antibody, the biggest advantage of this method is that they can collect EPCs with higher purity, but many scholars have found that the number of isolated CD34 + or CD133 + cells is very small, and they often do not proliferate when cultured alone, and can only be used with CD34. -Or mature endothelial cells have proliferative activity only when co-cultured [2]. Moreover, immunomagnetic bead sorting has the disadvantages of complicated operation and high cost, so it is difficult to promote. CD34 has been used by some researchers as a specificity for the isolation of endothelial progenitor cells. Marker [4]. CD34 is highly expressed in early hematopoietic stem cells and decreases with maturation of hematopoietic cells, but it is also expressed in vascular endothelial cells. Endothelial cells are difficult to distinguish between mature endothelial cells and endothelial progenitor cells with vascular wall detachment. In recent years, CD133 has been reported to be a five-time transmembrane cell surface molecule (Mr120000), an early antigen, which is selectively in the bone marrow and peripheral Hematopoietic stem cells and endothelial progenitor cells are expressed [5], but not on mature endothelial cells [4]. Therefore, CD133 sorting can be used to exclude mature endothelial cells from the vascular wall.
During embryonic development, vascular endothelial cells are present in various organ tissues. With the functional differentiation of each organ, vascular endothelial cells develop into organ-specific endothelial cells, but endothelial cells also have common antigens. In order to identify adherent spindle cells They are endothelial cells, and we use endothelial cell-specific markers KDR, CD133, and CD34 to identify them. The results show that adherent spindle cells express these specific markers to varying degrees, which indicates that the cultured spindle cells are endothelial cells.

Umbilical cord blood, as a stem cell resource, has many advantages such as non-trauma, rich resources, and early stage stem cells. The number of hematopoietic stem progenitor cells in cord blood is significantly higher than that of peripheral blood, which is one of the sources of hematopoietic stem cells. Some people estimate that the ratio of CD34 + cells in bone marrow, cord blood, and adult peripheral blood is 3: 2: 0.2. We isolated CD133 cells from cord blood and successfully induced differentiation into endothelial cells, suggesting that endothelial progenitor cells are present in cord blood, which can be used as Source of endothelial cells However, the endothelial progenitor cells contained in each cord blood are limited. Therefore, in order to meet the requirements of therapeutic vascular reconstruction, it is necessary to further explore the appropriate growth factor combination and time conditions so that it can be stably expanded in vitro. Make endothelial progenitor cells closer to the clinic in the treatment of ischemic diseases.