Experimental Study On HLA-G Expression Of Mesenchymal Stem Cells From Menstrual Blood And Umbilical Cord

Objective : Increasing incidence of autoimmune premature ovarian failure has gradually attracted attention,but its complex etiology leads to the absence of effective treatment options.Especially for young unfertile patients with premature ovarian failure,restoring fertility is the most urgent desire.Although current hormone replacement therapy(hormone replacement therapy,HRT)can relieve clinical symptoms and maintain a "young" state,it can not restore fertility.With the development of medical science,mesenchymal stem cells(mesenchymal stem cell,MSC)have become an alternative source of regenerative medical stem cells due to their pluripotency and powerful ability to regulate immune responses.Where human leucocyte antigen G(G)is an important mediator of MSC mediated immune regulation.Although many studies have demonstrated that HLA-G have strong immunosuppressive properties and can play an important role in many aspects,such as maternal immunity,autoimmune diseases and allograft.Nevertheless,there are a wide range of MSC sources,including bone marrow,umbilical cord,and trans-blood mesenchymal stem cells.Whether there are differences in HLA-G expression levels MSCs different sources has not been studied.Transblood-derived endometrial mesenchymal stem cells(Endometrial mesenchymal stem cells isolated from menstrual blood,MB-MSC)are relatively new types of stem cells,which are characterized by easy collection,non-invasive,no ethical problems and no autoimmune rejection.Human umbilical cord mesenchymal stem cells confirmed that human umbilical cord mesenchymal stem cells have great potential for allograft transplantation,and their relative immune immaturity,more primitive progenitor cells and a wide range of sources make it a hot spot in MSC transplantation and immunity.Therefore,MB-MSC 、 HUC-MSCs was chosen as the MSCs representative to compare the expression and function of HLA-G in order to further explore the treatment mechanism of immune premature ovarian failure.Methods: MB-MSC 、 HUC-MSCs were collected for culture,passage(2-3generations)and observation of cell morphology.After about 85% cell fusion,the expression of soluble HLA-G in culture medium was determined and compared by ELISA method.Through Western blot identification and comparison of expression HLA-G between different MSC from protein level The HLA-G;The HLA-G of MB-MSC localization by immunohistochemistry.CCK8 method was used to detect the proliferation of lymphocytes after co-cultured with lymphocytes in different concentration gradient MB-MSC to study the mechanism of MB-MSC and HLA-G.Results:1.observe the cell morphology,HUC-MSCs subculture 4 hours can normal adherent growth,showing fibroblast-like,spindle-shaped morphology;MB-MSCs in inoculation 24 h visible adherent,scattered in petri dish growth,mainly fusiform,2generation culture about 2 d can reach 80%~90% fusion.2.ELISA results showed that both MB-MSC and HUC-MSC could secrete soluble HLA-G,but there was no significant difference in their expression levels(3.3±0.21 v.s.2.7±0.39,P=0.139).3.Western Blot results showed that both MB-MSC and HUC-MSC were expressed at protein level,and the expression level of MB-MSC was significantly higher than that of HUC-MSC(p <0.05).4.cell immunohistochemical results showed that HLA-G expression was strongly positive in MB-MSC cytoplasm.5.The proliferation state of lymphocytes and MB-MSC was detected by CCK8method: the inhibition degree of MB-MSC with different concentration gradient on lymphocyte proliferation was(71.67±1.43,60.95±0.42,59.25±2.83,44.05±1.62,30.77 ± 0.68,29.77 ± 0.59),the difference was statistically significant(P=0.007);The difference of lymph inhibition rate between 4×104 and1.25×103 was statistically significant(71.67±1.43 v.s.29.77±0.59,P=0.015).After the addition of HLA-G antibody,the difference of lymphocyte inhibition rate between the two groups was statistically significant(71.67±1.43 v.s.28.65±0.77,P <0.05).Conclusion:1.MB-MSCs and HUC-MSCs can secrete soluble HLA-G,and there is no significant difference in secretion.2.MB-MSCs are expressed more HLA-G than HUC-MSC at the protein level3.HLA-G expressed in MB-MSCs’ cytoplasm.4.MB-MSC can inhibit lymphocyte proliferation and have concentration dependence,that is,the more cells number,the stronger the inhibition of lymphocyte proliferation;MB-MSC mainly through HLA-G play an inhibitory effect on lymphocytes,to provide experimental basis for stem cells to treat immune premature ovarian failure and other immune diseases.