Compare With Two Isolate Processes For UCMSCs And The Study On Immunoregulation Of UCMSCs On UCB CD4~+T Lymphocytes

Umbilical cord mesenchymal stem cells (UCMSCs)are a kind of adult stem cells with self-renew and multilineage differentiation. They have the function of immune regulation, hematopoietic support and tissue repair properties. The study on isolation method and immune function of MSCs have great significance for clinical application on autoimmune disease and graft-versus-host disease(GVHD). This research include three parts.First we compared with two isolate processes for MSCs from umbilical cord, to distinguish two general approaches.The mesenchymal stem cells (MSCs) were isolated from umbilical cordby tissue explants adherent method or enzymatic digestionmethod and expanding cultured in vitro. Cell morphous was observed with an inverted microsco, surface markers were detected with flow cytometry, growth curves of cells were drawn and multi-differentiation capacitywasidentified by osteogenic differentiation respectively.Research indicates that the isolation of UCMSCs by both of the two methods were efficient.There were no significant difference in surface markers and osteogenic differentiation with two methods, but the tissue explants adherent method has a certain advantage in the success rate of cell isolation proliferation ability and experimental costs.We can conclued that isolation of UCMSCs with the tissue explants adherent methodare more in line with the requirements of standardization and scale, and therefore more worthy of promotion.Second part is the immunomodulatory study of UCMSCs on alien UCB (Umbilical cord blood) CD4+T lymphocytes proliferation and differentiation. Direct contact coculture or transwell coculture system of UCMSCs and phytohaemagglutinin (PHA)-activated UCB CD4+T lymphocytes with different proportion was constructed. The proliferation of lymphocyte was measured by blood cell counter, percentage of CD4+CD25+/CD4+ were detected with flow cytometry, relative expression amount changes of markergene Foxp3 in regulatory T cell were observed with fluorescencequantitative PCR.The study found that the UCMSCs could inhibit evidently the proliferation of PHA-activated UCB CD4+T lymphocytes, increases the percentageof CD4+CD25+ regulatory T cells in CD4+T lymphocytes and the relative expression amount with gene Foxp3. These effects were positively correlated with the number of UCMSCs and the effect of direct contact coculture is stronger than that of transwell coculture.The UCMSCs could inhibit the proliferation of PHA-activated UCB CD4+T lymphocytes and change the percentage of regulatory T cell subsets. These results indicate that UCMSCs might negatively modulate UCB CD4+T lymphocytes-mediated immunity effects.The last part is the study on effect of UCMSCs on alien UCB (Umbilical cord blood) CD4+T lymphocytes apoptosis. Direct contact coculture or transwell coculture system of UCMSCs and dexamethasone-stimulated UCB CD4+T lymphocytes was constructed. CD4+T lymphocytesapoptosis were detected by flow cytometry.Research shows that the UCMSCs could inhibit evidently the dexamethasone-induced apoptosis of UCB CD4+T lymphocytes, increases the percentage of living cell in UCB CD4+T lymphocytes. The effect of direct contact coculture is stronger than that of transwell coculture.We can conclued that the UCMSCs could inhibit the dexamethasone-induced apoptosis of UCB CD4+T lymphocytes and increases the percentageof living cell.