Effects Of MiR-17 On Immunomodulatory Properties Of Human Periodontal Ligament Stem Cells

Objective:The immunomodulatory properties of hPDLSCs and its role in immune regulation were studied,the effect of miR-17 on the immunoregulation characteristics,proliferation and apoptosis of hPDLSCs is further explored,which lays a theoretical foundation for the development and treatment of periodontitis.Methods:(1)HPDLSCs was obtained by cloned culture with the limiting dilution method.Colony forming assay and flow cytometry were used to detect the proliferation ability of hPDLSCs;the immunofluorescent cytochemistry was used to detect the surface molecules CD146 and STRO-1 of mesenchymal stem cells;flow cytometry was used to detect the surface markers of mesenchymal stem cells(CD90,CD13 and CD90),hematopoietic stem cell surface markers(CD45 and CD34),and endothelial cell surface marker(CD31).The multidirectional differentiation ability of hPDLSCs was detected by osteogenic and adipogenic induction in vitro.(2)Establish a co-culture system of hPDLSCs and PBMCs.The experimental group was divided into the control group and the co-culture group.The expression of level the immunomodulatory factors and inflammatory factors in the co-culture system were detected by qRT-PCR and Elisa,so as to explore whether the cultured hPDLSCs had immunological performance.The factors to be detected are as follows:TGF-?,IDO,IL-10,TNF-?,IFN-?,IL-1?.(3)The mimics(pre-miR-17)and inhibitor(anti-miR-17)were transfected into hPDLSCs,and the Transwell co culture system was established with activated PBMCs respectively.The expression of level of the immunomodulatory factors(TGF-?,IDO,IL-10)in the co-culture system were detected by three methods,and these three methods are qRT-PCR,Western blot and Elisa,respectively.The effect of miR-17 on the secretion of soluble immunoregulatory factor after co culture was investigated.Secondly,the cycle and apoptosis of hPDLSCs in each group were detected by flow cytometry,and the effect of miR-17 on proliferation and apoptosis of hPDLSCs was explored.Results:(1)HPDLSCs,which was cloned by the limiting dilution method,was long shaped fusiform or irregular,and the cells were arranged in vortexes and radiates;Clonogenic assay and cell cycle detection showed that hPDLSCs had high proliferative capacity;immunofluorescence analysis cell surface molecules showed that hPDLSCs positive expression of mesenchymal stem cell surface markers STRO-1,CD 146,flow cytometry analysis of cell surface molecules showed hPDLSCs high expression of mesenchymal stem cell surface markers CD90,CD 13,CD44,low expression of hematopoietic stem cell surface markers CD34,CD45,almost non expression The surface marker of skin cell CD31;The mineralized nodules can be seen after osteogenesis induced,and the Alizarin red staining was positive.Lipid droplet was observed after lipogenic induction induction,and the oil red O staining was positive.(2)After establishing the co-culture system of hPDLSCs and PBMCs.The changes of immunomodulatory factors in hPDLSCs(TGF-??IDO?IL-10)and inflammatory factors in PBMCs(TNF-??IFN-??IL-1?)were detected by qRT-PCR and Elisa respectively.Compared with the control group,the expressions of mRNA and protein in immunomodulatory factors were significantly increased(P<0.01),while the expressions of inflammatory factors were decreased(P<0.01 or P<0.05).(3)After transfecting mimics(pre-miR-17),inhibitor(anti-miR-17)and unrelated sequences to hPDLSCs respectively,the miR-17 expression after 48h was detected by qRT-PCR,and there was no statistical difference between the blank control group and the unrelated sequence control group(P>0.05),and the interference of the transfection reagent to this experiment.In addition,the transfected hPDLSCs was used to establish a Transwell co-culture system with activated PBMCs respectively.After co-culture of 48h,the expression of the immunomodulatory factors(TGF-?,IDO,IL-10)in the co-culture system was detected by three methods of qRT-PCR,Western blot and Elisa,and the expression group and the over expression control group were found.Compared with the control group,the expression of immunoregulatory factors increased significantly(P<0.01 or P<0.05);while the expression of these factors in the inhibitory group was lower than that in the control group(P<0.01 or P<0.05).(4)After 48h,the proliferation and apoptosis of hPDLSCs were detected by flow cytometry.It was found that the proliferation index increased(P<0.01)and the apoptosis rate decreased(P<0.01)in the overexpression group compared with the overexpression control group.And the proliferation index decreased(P<0.01)and the rate of apoptosis increased(P<0.05)in the inhibition group compared with the inhibition control group.Conclusions:(1)The hPDLSCs with limited dilution polyclonal method has stronger self renewal ability and multidirectional differentiation potential of stem cells.(2)hPDLSCs has a significant immunomodulatory effect and can play an immunoregulatory role by secreting soluble immunoregulatory factors.(3)miR-17 can regulate the immunomodulatory effect of hPDLSCs.It is involved in immune regulation by promoting hPDLSCs secretion of soluble immunoregulatory factors.(4)miR-17 can regulate the proliferation and apoptosis of hPDLSCs,and it can promote proliferation and inhibit apoptosis of hPDLSCs.