Mesenchymal Stem Cells Ameliorate Premature Ovarian Failure Via Regulatory T Cell Induction

OBJECTIVEAutoimmune ovarian disease has been recognized as one of the important causes of premature ovarian failure(POF).The abnormal expression of regulatory T cell(Treg)and related cytokines may involve in the pathogenesis of POF.Mesenchymal stem cells(MSCs)from different sources could repair damaged ovarian function in the animal models.However,whether the immune modulatory mechanisms play a role in the effect of MSCs on POF,there have been no reports.The present study will innovatively study the repair effect of MSCs on ovarian failure from the view of immune regulation.The present study will validate that whether MSCs repair damaged ovarian function via regulation of Treg and related cytokines.This study will provide a strong theoretical basis for the mechanisms of MSCs therapy for POF and clinical guideline for MSCs therapy in POF.METHODSThe first chapter:The blood samples were collected from women with different ovarian function.The expression of estradiol(E2)and follicle stimulating hormone(FSH)were examined by chemoluminescence method.The proportion of CD4+Foxp3+Treg was detected via flow cytometry(FCM).The expression of cytokines including interferon-γ(IFN-γ),transforming growth factor β1(TGF-β1)and interleukin 10(IL-10)were determined through enzyme-linked immuno sorbent assay(ELISA).The second chapter:Autoimmune POF mouse models were established by immunization with a murine zona pellucida 3 peptide in Freund’s adjuvant,and were verified via vaginal smears,serum sex hormone,morphology of ovaries,antibody to ZP3(AZP3Ab)and CD45 in the ovarian tissue.The proportion of CD4+CD25+Foxp3+Treg in the spleen,the expression of Foxp3 in the ovary,and the expression of cytokines(IFN-γ,TGF-β1 and IL-10)in the ovary were determined.The third chapter:Under the condition of cell-cell contacts or transwell,bone marrow mesenchymal stem celss(BMSCs)were co-cultured with actived splenocyte for 96h,and neutralizing antibody to TGF-β1 or the prostaglandin E2(PGE2)inhibitor was added when necessary.The proliferation of CD4+ T cells,the proportion of Treg and the expression of cytokines(IFN-y,TGF-β1 and IL-10)were determined.Furthermore,CD4+CD25-T cells were sorted bymagnetic beads and were co-cultured with BMSCs for 96h.The proportion of CD4+CD25+ T cells and the expression of cytokines(IFN-y,TGF-β1 and IL-10)were determined.The fourth chapter:After BMSCs transplants into POF mouse models via intravenous injection or ovarian situ injection,in vivo tracking,ovarian endocrine functions,immune function and fertility were examined.The test items of the ovarian endocrine functions included serum sex hormone,morphology of ovary,and antibody to anti-mullerian hormone(AMH in the ovarian tissue.The test items of immune function included the proportion of CD4+CD25+Treg in the slpeen,the expression of Foxp3,IFN-y,TGF-β1 and IL-10 in the ovaries.POF mouse models were mated with male mice and were checked for pregnant four weeks after cell transplantation.RESULTSThe first chapter:Compared with their healthy counterparts,women with POF had lower serum E2 level,higher serum FSH level,smaller proportion of CD4+Foxp3+Treg,lower expression of TGF-β1 and IL-10,and higher expression of IFN-y.The second chapter:Compared with the control group,POF mouse models had more proportion of estrous cycle dysfunction,lower serum E2 level,higher serum FSH level,less growing ovarian follicle,higher expression of AZP3Ab and CD45 in the ovary,lower expression of Foxp3+ in the ovary,smaller proportion of CD4+CD25+Foxp3+Treg in the spleen,lower expression of TGF-β1 and IL-10 in the peripheral blood,and higher expression of IFN-y in the peripheral blood.The third chapter:Under the condition of cell-cell contacts,high dose of BMSCs could inhibit the proliferation of CD4+ T cells,induce the production of Treg and reverse the expression of cytokines(IFN-y,TGF-β1 and IL-10).Compared with the condition of cell-cell contacts,the co-culture of BMSCs and actived splenocyte of POF mouse models displayed stronger immune suppression.And the neutralizing antibody to TGF-β1 or the prostaglandin E2(PGE2)inhibitor could reverse these immune suppression effects.Moreover,high dose of BMSCs could induce CD4+CD25-T cells to differentiate into CD4+CD25+ T cells,accompanied with the increased expression of IFN-γand TGF-β1 and the decreased expression of IFN-y.And these effects were dose dependent.The fourth chapter:BMSCs transplants can migrate into the interstitial tissues of ovaries,but not into the follicles.The ovarian endocrine function,fertility and immune function of POF mouse models were improved.As for the ovarian endocrine function,serum estrogen level improved,serum FSH decreased,each stage follicles increased,and the expression of AMH in the ovary tissue increased.As for the immune function,the proportion of CD4+CD25+Treg in the spleen increased,the expression of Foxp3,TGF-β1 and IL-10 in the ovary increased,while the expression of IFN-yin the ovary decreased.As for the fertility,the pregnancy rate and average little size were improved.CONCLUSIONBMSCs could repair damaged ovarian function via inducing the production of Treg,TGF-β1 and IL-10 and the inhibition of IFN-y.