Effect Of Extracellular Vesicles From Mouse Adipose-derived Mesenchymal Stem Cells On The Conversion Of CD4~+T Cells To Treg Cells

Objective:This experiment establishes a co-culture system of ADSC-EV and CD4~+T cells in vitro.Flow cytometry detects the change of the proportion of regulatory T cells(Tregs)after co-culture of ADSC-EV and CD4~+T cells,and then explores the immune regulatory function of ADSC-EV and its possible immunoregulatory mechanism,which lays an experimental foundation for non-cellular treatment of autoimmune diseases and inflammatory diseases based on ADSC-EV.Method:Primary mouse ADSCs were extracted and cultured by type I collagenase digestion,and the expression of surface-specific markers CD29,Scal-1,CD34 and CD45was detected.Alizarin red,oil red O,toluidine blue staining was used to detect their ability to differentiate into osteocytes,chondrocytes and adipocytes.Immunomagnetic bead negative sorting was used to extract CD4~+T cells from mouse spleen.The supernatant of P3-P6 cells was collected,and extracellular vesicles were separated by ultracentrifugation.The morphology of extracellular vesicles was detected by transmission electron microscopy,and the marker proteins CD9 and CD63 of extracellular vesicles were identified by Western Blot method.The cell co-culture experiment was divided into four groups:(1)blank control group:culturing CD4~+T cells alone;(2)10ul extracellular vesicle intervention group:10ul EV+CD4~+T cells;(3)20ul extracellular vesicle intervention group:20ul EV+CD4~+T cells;(4)positive control group:tacrolimus+CD4~+T cells.The CD4~+T cell suspension of each group was recovered after 72 h,and the proportion of Treg cells was detected by flow cytometry.The proportion of Treg cells was expressed as x±s.SPSS22.0 statistical software was used to compare the blank control group,10ul EV intervention group,20 ul EV intervention group and positive control group by analysis of variance(LSD-t).P<0.05was statistically significant.Result:ADSCs extracted from mouse fat met the international criteria for the identification of mesenchymal stem cells.The extracellular vesicles of adipose-derived mesenchymal stem cells in mice were double-membrane-encapsulated vesicles with typical protein markers under transmission electron microscopy.The percentage of CD4~+CD25~+Foxp3~+T cells in the blank control group was 8.25±0.16,the percentage of CD4~+CD25~+Foxp3~+T cells in the 10ul EV intervention group was 10.11±0.44,the percentage of CD4~+CD25~+Foxp3~+T cells in the 20ul EV intervention group was10.32±0.78,and the percentage of CD4~+CD25~+Foxp3~+T cells in the positive control group was 12.90±0.77.The difference between 10ul EV intervention group and 20ul EV intervention group was statistically significant compared with blank control group(P<0.05),while the difference between 10ul EV intervention group and 20ul EV intervention group was not statistically significant(P>0.05).Extracellular vesicles play an immunoregulatory role by increasing the proportion of Treg cells.Conclusion:Mouse adipose-derived mesenchymal stem cells can secrete extracellular vesicles,and derived extracellular vesicles can stimulate the conversion of CD4+T cells into Treg cells,thereby playing a regulatory immune function,enriching research methods in the field of non-cellular immunomodulatory therapy.