Bone Marrow Mesenchymal Stem Cell Therapy For Autoimmune Premature Ovarian Insufficiency

Background and ObjectivePremature ovarian insufficiency(POI)is a common gynecologic endocrine disease.The etiology of POI is heterogeneous,involving genetic,autoimmune,metabolic,environmental and iatrogenic factors.Autoimmune POI is so far regarded as a condition in which ovaries are attacked by lymphocytes or anti-oocyte antibodies,caused by dysregulation of immune system.And because of complex involvement of immunomodulatory disorders,there hasn't been a systematic framework of its underlying mechanism,which leads to a lack of effective methods of etiological therapy.Hormone replacement therapy(HRT)is nowadays the main strategy.Recently,application of mesenchymal stem cells(MSCs)is much valued.There have been animal and clinical studies of different kinds of MSC therapy for idiopathic POI or chemotherapeutic POI,while bone marrow mesenchymal stem cells(BMSCs)therapy for autoimmune POI are rarely studied.Thus,in this study,we aimed to investigate the therapeutic effects of BMSCs in autoimmune POI,both in vitro and in vivo.We will also probed the potential signal pathways of the therapeutic effects.MethodIn Chapter 1,effects of IFN-y at different concentrations(0,10,20,50,100 ng/ml respectively)in granulosa cell morphology,proliferation and apoptosis were tested and analyzed.The minimal effective doze was chosen to be the optimum concentration.Levels of hormone synthesis and inflammatory factors were investigated at the optimum concentration.In Chapter 2,we evaluated the potential mechanical effects of IFN-y-induced autoimmune damage in granulosa cells on two pathways,namely,the TLR4 recognization process and the consequent activation of NLRP3 inflammasome-induced pyropsis.In Chapter 3,by co-culturing BMSCs and IFN-y-induced granulosa cells using transwells,we explored the therapeutic effects of BMSCs on this cell model.Cell proliferation,apoptosis,steroidogenesis and NLRP3 inflammasome-induced pyroptosis were investigated in different groups.In Chapter 4,by in situ injection of BMSCs in ovaries of autoimmune POI mice,we investigated the therapeutic effects of BMSCs on autoimmune POI mice model.Irregular estrous cycle,serum E2 and FSH levels were tested.H&E staining,counts of follicles at various stages,TUNEL analysis were tested in ovaries of different groups.Result1.IFN-y could be used to induce autoimmune damage in granulosa cells,with the concentration of 50 ng/mL and co-cultured for 24 h as the optimum condition.2.The TLR4 recognization process and the canonical pyroptosis pathway were both up-regulated in IFN-y-induced autoimmune damage in granulosa cells.3.Co-culturing with BMSCs could reverse IFN-y-induced autoimmune damage in granulosa cells,presenting rescued proliferation and apoptosis rates,improved hormone synthesis ability,and down-regulated pyroptosis pathway results.4.BMSCs therapy in autoimmune POI mice model could reduce the proportion of irregular estrous cycle of mice,rescue serum E2 and FSH levels,increase the numbers of primordial and primary follicles,and reduce the number of atresic follicles,suppress granulosa cell apoptosis in ovaries.ConclusionIn this study,we proposed a method to establish an in vitro model for autoimmune POI using IFN-y to induce inflammation in granulosa cell line KGN,which could in a certain degree simulate the immunomodulatory disordered environment for granulosa cells in autoimmune POI.And both in vitro and in vivo,we proved the therapeutic effects of BMSCs on autoimmune POI.