| Objective In this experiment,the co-culture system of CD146~+human adipose derived pericytes/perivascular cells(CD146~+hAD-PCs),bone marrow mesenchymal stem cells(BMSCs)and PHA-stimulated peripheral blood mononuclear cells(PMBC)were established in vitro to compare the immunomodulatory function of CD146~+hAD-PCs and BMSCs on allogeneic peripheral blood lymphocytes,it is hoped to further provide experimental basis and new ideas for the clinical application of CD146~+hAD-PCs in the treatment of graft versus host disease(GVHD)and to solve the problem of limited sources of BMSCs in the future.Methods 1.CD146~+hAD-PCs were isolated from fresh adipose tissue by enzymatic digestion and multiparameter flow cytometry and cultured.2.Human peripheral blood mononuclear cells(PBMC)were isolated by density gradient centrifugation.3.CD146~+hAD-PCs and BMSCs with PHA-stimulated PBMC were cultured for 72 hours.The experiment was divided into experimental groups(CD146~+hAD-PCs+PBMC+PHA group and BMSCs+PBMC+PHA group),positive control group(PBMC+PHA group)and negative control group(PBMC group alone),to compare the immunomodulatory function of the two derived stromal cells on peripheral blood lymphocytes.4.The proliferation of PBMC under coculture conditions was determined by CCK-8 assay.5.Apoptosis of PBMC cells in each culture group was determined by flow cytometry.6.Flow cytometry was used to detect the proportion of Treg,Th1,Th2,Tc1 and Tc2 cells in each experimental group and the positive control group.7.ELISA measured IL-2,IL-10,TNF-α,and IFN-γin the supernatants of each culture group.8.The m RNA levels expression of IFN-γ,T-bet and GATA3 in T lymphocytes of each group were determined by RT-q PCR.Results 1.Detection of allogeneic lymphocyte proliferation under co-culture conditions by CCK-8 method:CD146~+hAD-PCs+PBMC+PHA group(experimental group),and BMSCs+PBMC+PHA group(experimental group)were found to be compared with the PBMC+PHA(positive control group)and PBMC groups,respectively,CD146~+hAD-PCs and BMSCs both had inhibitory effects on lymphocyte proliferation stimulated by the addition of PHA,and the different proportions of the two stromal cells were proportional to their ability to inhibit PBMC proliferation in the mixed culture system with PBMC,and there was a dose-dependent relationship,but there was no statistical difference between the same proportions of the two.2.FCM detection of apoptosis:compared with the PBMC group,it was found that the other groups had statistical differences with it,which confirmed that some lymphocytes had apoptosis after the action of stromal cells.3.FCM detected the changes of T lymphocyte subsets:The effects of CD146~+hAD-PCs and BMSCs on peripheral blood T lymphocyte subsets were compared with PBMC+PHA group.The former could promote the increase of the proportion of Treg,Th2,Tc2 cell subsets in PBMC stimulated by PHA,while the proportion of Th1,Tc1 cell subsets decreased.However,there were no statistically significant difference in the effects of CD146~+hAD-PCs and BMSCs on T lymphocyte subsets.4.ELISA showed decrease of IL-2,TNF-α,IFN-γ,and increase of IL-10 in the supernatants compared with the PBMC+PHA group.5.q RT-PCR showed that T lymphocytes showed decreased expression of IFN-γand T-bet and increased GATA3 expression level,which was statistical different.Conclusion Compared with human BMSCs,CD146~+hAD-PCs increased the proportion of Treg,Th2,and Tc2 cell subsets in PHA-stimulated lymphocytes and promoted their IL-10secretion in vitro,which down regulated the proportion of Th1 and Tc1 cell subsets and inhibited the secretion of IL-2,TNF-α,and IFN-γ.Therefore,CD146~+hAD-PCs,have similar inhibition to BMSCs inhibiting PHA-stimulated allolymphocyte proliferation,as well as immunomodulatory effects. |
