The Effects Of Culture Generation On The Activity Of Menstrual-derived Endometrial Stem Cells And Its Improving Symptoms Of Type 1 Diabetes

BackgroundType 1 diabetes(T1DM)is a chronic autoimmune disease characterized by causing insulin deficiency and hyperglycemia.At present,the main clinical application is insulin,but it cannot control the severe symptoms of T1DM.Recently,immunotherapy,pancreas or Langerhans cell transplantation,and cell therapy have received more and more attention and recognition.Mesenchymal stem cells(MSC)not only have the potential for self-renewal and multi-directional differentiation,but also have strong immune regulation,among them,menstrual blood derived endometrial stem cells(MenSCs)have the advantages of rich sources,strong value-added activities,and non-invasive isolation and acquisition methods,therefore,MenSCs is the research focus of stem cell therapy.Objective1.Identify the biological characteristics of MenSC scultured in vitro at different passages(Passage 3,9 and 15),and select the optimal MenSCs as the research target for the treatment of T1DM;2.Clarify the effect of improving the symptoms of T1DM after intravenous infusion of MenSCs and UcMSCs.Methods1.Density gradient centrifugation to separate and extract MenSCs,analyze surface markers of MenSCs by flow cytometry,and verify the differentiation of adipogenic,osteogenic and chondrogenic three lines;2.MenSCs were cultured to P3,P9 and P15 generations,and observe the cell morphology by calcein-AM staining;?-galactosidase staining to detect the senescence of MenSCs in different culture passages;active oxygen kit to detect MenSCs Oxygen content;3.Establish co-cultivation system of PBMCs with P3,P9 and P15 generation MenSCs.MTT method was used to detect the proliferation activity of PBMCs;PI staining was used to detect the apoptosis and cell cycle of PBMCs;detect the effects of MenSCs on CD3~+and CD19~+subpopulations of PBMCs by flow cytometry;4.Low-dose multiple intraperitoneal STZ injections to establish a T1DM mouse model.After intravenous infusion of MenSCs and UcMSCs,monitor changes in fasting blood glucose,body weight and food consumption of the mice;5.64 days after MenSCs and UcMSCs transplantation,blood samples from mice were collected for biochemical detection,and protein microchips were used to detect mouse peripheral blood cytokines.Weigh the kidney,liver,and spleen to calculate the organ index,and perform HE staining on the pancreas,kidney,liver,and spleen.Results1.The extracted MenSCs have a typical fibroblast-like structure,which positively expresses CD73,CD90,CD105,and HLA-ABC,and negatively expresses HLA-DR,and can differentiate into adipogenic,osteogenic and chondrogenic cells in related induction medium;With the increase of culture passages,the area of MenSCs cells increased significantly,and a large number of filamentous pseudopods began to appear in P15MenSCs.The degree of senescence and the content of reactive oxygen species increased significantly with the increase of culture passages;2.In the co-culture system of MenSCs and PBMCs,MenSCs significantly increased the activity of mouse spleen lymphocytes and reduced lymphocyte mortality;And with the increase of culture generation,MenSCs significantly reduced the ability to promote lymphocyte survival and reduce death;further cell cycle tests found that MenSCs did not stimulate lymphocyte proliferation and division activity,but could significantly reduce lymphocyte death and fragmentation,and cultured The increase of generations can significantly reduce the ability of MenSCs to maintain lymphocyte survival.In addition,MenSCs of different culture generations have no significant effect on the percentage of CD3 and CD19 cell subsets in mouse spleen lymphocytes in vitro;3.After transplantation of MenSCs and UcMSCs,both can significantly improve fasting blood glucose in mice,and both trends are consistent;further pancreatic section results also showed that the number and area of pancreatic islets in the pancreas of the stem cell treated group were significantly higher than those of the control group;UcMSCs significantly increased body weight and food intake of experimental animals,while the MenSCs treated groups were not significantly different from the control group;protein chip results show that stem cell transplantation can significantly reduce TNF-?,IFN-?and increase IL-6 and VEGF levels in mice.HE staining and biochemical indicators also show that stem cell transplantation can significantly improve liver and kidney function in experimental animals;while there was no significant improvement on the spleen.Conclusion1.With the increase of culture passages,the cell biological activity of MenSCsdecreases significantly;2.MenSCs can systematically improve the T1DM experimental animal relatedsymptoms.