Experimental Study Of Adipose-derived Mesenchymal Stem Cells Regulating Tim-3 And Treg Cells In The Treatment Of Premature Ovarian Failure

Objective:Autoimmune disorders are one of the important causes of premature ovarian failure?POF?,which can be manifested as abnormalities in peripheral blood regulatory T cells?Treg?and related factors.Studies have found that the use of adipose-derived mesenchymal stem cells?ADSCs?to repair ovarian function in immune POF mouse models can promote Treg cell differentiation and proliferation.Tim-3 is involved in the differentiation and immunosuppressive functions of Treg cells,but it has not been reported whether it is involved in the pathogenesis of POF.This study explored the regulation of Tim-3 on Treg cells and related factors in POF,and its role in the repair of ovaries in a POF model by ADSCs.Method:1)enzyme-linked immunosorbent assay?ELISA?was used for detecting the level of TGF-?1,IFN-?and Tim-3 in POF group,ovarian insufficiency group and control group;2)Flow cytometry was used to detect the proportion of Treg cells and the expression of Tim-3 on Treg cells in peripheral blood;3)Immune POF mouse model was constructed by using morine ZP3,and were verified via vaginal smears,the expression of ZP3;4)POF mice were transplanted with ADSCs.The ELISA was used for detecting the changes in hormone levels?E₂ and FHS?and cytokines?TGF-?1 and IFN-??in normal,POF and stem cell groups;5)Immunohistochemistry was used to detect the expression of Tim-3 in mouse ovary;6)Flow cytometry was used to detect the ratio of Tim3⁺CD4⁺CD25⁺Treg cells in spleen of mice;7)Spearman correlation analysis was used to analyze the correlation between Tim-3 levels,Tim3⁺CD4⁺CD25⁺Treg cells and related cytokines.Results:1)ELISA test found that compared with control group,serum E₂,AMH and Tim-3 levels in patients with POF group and ovarian dysfunction group were significantly reduced and FSH levels were increased.TGF-?1 expression was down-regulated,while IFN-?was up-regulated?P<0.05?;2)Flow cytometry showed that with the decline of ovarian reserve function,the proportion of peripheral blood CD4⁺CD25⁺Treg cells and Tim3⁺CD4⁺CD25⁺Treg cells in the three groups of patients also decreased.It has a positive correlation with the TGF-?1 level and a negative correlation with the IFN-?in peripheral blood;3)Compared with the control group,the estrous cycle of the mice in the POF group was disordered,serum ZP3 and FSH levels were significantly increased,E₂ levels were reduced,structural and functional changes in the ovary,so the model was successfully constructed;4)After ADSCs transplantation,serum levels of E₂ and TGF-?1 in mice increased,and levels of FSH and IFN-?decreased?P<0.05?;5)Immunohistochemical detection showed that compared with the control group,the ovarian Tim-3 level in the POF group was reduced,then the expression was increased after ADSCs transplantation;6)Flow cytometry revealed that compared with the control group,the proportion of Tim3⁺CD4⁺CD25⁺Treg cells in peripheral blood and spleen of POF group mice was significantly reduced?P<0.05?,and the proportion was increased after s ADSCs transplantation;7)Spearman correlation analysis showed that Tim-3 expression in mouse ovary was positively correlated with the proportion of Tim3⁺CD4⁺CD25⁺Treg in the spleen?r=0.728,P<0.01?,positively correlated with serum TGF-?content?r=0.700,P<0.01?,and negatively correlated with serum IFN-?content?r=-0.673,P<0.01?.Conclusion:Decreased expression of Tim-3can reduce the proportion of Tim3⁺CD4⁺CD25⁺Treg cells,impaired the Treg cells,and enhanced the immune response,leading to ovarian inflammation changes,which may be the pathogenesis of POF.Decreased in Treg cells also reduces the level of TGF-?1 and increases the level of IFN-?,which leads to ovarian developmental disorders and excessive atresia,further promoting the development of POF.ADSCs can promote the expression of Tim-3,induce the differentiation and proliferation of Treg cells in the body,increase the level of TGF-?1,and inhibit the secretion of IFN-?,thereby improving ovarian reserve function,can provide a new experimental basis and clinical for the treatment of POF.