Effects Of SiFasL On Immunomodulatory Properties And Osteogenesis Of Human Periodontal Ligament Stem Cells

Objective:By using human periodontal ligament stem cells(cells,hPDLSCs)as the research object,the immunomodulatory properties were analyzed,and the immunoregulatory effect of small interfering RNA(siFasL)on hPDLSCs was investigated,and whether the low expression of FasL inhibited the proliferation of hPDLSCs.The effects of death and osteogenesis have a theoretical basis for studying the pathological mechanism of periodontitis and clinical effective treatment.Methods:(1)Primary hPDLSCs were obtained by enzymatic hydrolysis tissue mass method,and were isolated and purified by finite dilution method.The proliferation ability of hPDLSCs was detected by clonal formation assay and flow cytometry.CD146 and STRO-1 were detected by immunofluorescence cytochemistry.Flow cytometry was used to detect the expression of mesenchymal stem cell surface markers(CD90,CD 105,CD44)and hematopoietic stem cell surface markers(CD45,CD34).The osteogenic induction of hPDLSCs was performed in vitro,respectively,and fat induction was performed to test whether it has the ability to multi-directionally differentiate.(2)Mononuclear cells in human peripheral blood were placed in the upper layer,hPDLSCs cells were placed in the lower layer,and two cells were co-cultured through Transwell chamber,and then the experiment was divided into non-co-culture group and co-culture group.qRT-PCR,Elisa and Western blot were used to detect the mRNA and protein expression of inflammatory factors(VEGF)in hPDLSCs in non-co-cultured and co-cultured groups,and to explore the immunoregulation of hPDLSCs.performance.(3)Construction of siFasL transfection system,transfection hPDLSCs,divided into blank control group,independent sequence transfection group and transfection siFasL group,and then co-culture with PBMCs.To explore the immunomodulatory effect of FasL on hPDLSCs in co-culture system,the expression of hPDLSCs immunomodulatory factor(TGF-??IDO?IL-10)mRNA and protein,and the inflammatory factors(TNF-??IFN-??IL-1?)mRNA and protein in PBMCs were detected by qRT-PCR,Western blot and Elisa,respectively.Secondly,by flow cytometry instrument testing transfection irrelevant sequence and transfection siFasL after each hPDLSCs cell cycle and cell apoptosis,explore siFasL influence on hPDLSCs proliferation and apoptosis,as well as the qRT PCR,Western blot detection Runx2 osteogenesis related gene and the expression of ALP in each group,as well as in vitro and osteogenesis after induction of alizarin red and ALP staining,explore siFasL effects on hPDLSCs osteogenesis characteristicsResults:(1)Enzymatic tissue block method combined with limiting dilution cloning culture to obtain hPDLSCs with long spindle-shaped or irregular radial or spiral arrangement;detection of proliferation of hPDLSCs:colony formation assay and cell cycle detection Both showed strong proliferative ability;hPDLSCs obtained by immunofluorescence assay positively expressed mesenchyinal stem cell surface markers STRO-1 and CD 146;the obtained hPDLSCs were analyzed by flow cytometry to obtain mesenchymal stem cell surface markers.The expression level of the substance is high,and the expression of surface markers and CD45 of hematopoietic stem cells is low.The obtained hPDLSCs were induced by in vitro osteogenic induction and stained with alizarin red.The result was(+).At the same time,the obtained hPDLSCs were induced to induce fat in vitro,and the results showed that oil red O staining was also observed in the naked eye(+).Under the microscope,a large volume of round lipid droplets in the cytoplasm of the cells can be seen.(2)HPDLSCs were co-cultured with PBMCs through Transwell chamber,using qRT-PCR technique for immunoregulatory factors(TGF-?,IDO,IL-10)in hPDLSCs and inflammatory factors(TNF-?,IFN-?)in upper cell PBMCs.The level of change in mRNA of IL-la)was detected,and Elisa detected the level of change in the protein concentration of the above cytokine in the cell culture supernatant of each of the two cells.The above experimental results showed that the mRNA and protein expression levels of immunoregulatory factors in hPDLSCs were significantly increased in the co-culture group compared with the non-co-culture group(P<0.01),and the amount and protein of inflammatory factors mRNA in PBMCs.The expression levels were all decreased(P<0.01).(3)The experimental design was:blank control sequence,interference-free control sequence,and siFasL101,siFasL102,siFasL103 five groups.The sequences of each group were transfected into hPDLSCs respectively,and transfected by real-time PCR 2 days after transfection.The mRNA expression level of FasL on hPDLSCs was determined.The results showed that there was no difference between the expression levels of the blank control group and the unrelated sequence control group(P>0.05),so the interference generated by the transfection reagent lipo2000 could be neglected.The mRNA expression levels of FasL in hPDLSCs were significantly decreased in the three groups of siFasL101,siFasL102 and siFasL103,and the difference was statistically significant(P<0.01).The optimal silencing sequence siFasL102 was screened out.Six hours after transfection,the hPDLSCs group transfected with irrelevant sequence and transfected with siFasL were co-cultured with CoA-activated PBMCs.After 2 days of co-culture,immunomodulation of hPDLSCs in each experimental group was performed using Elisa.The expression levels of mRNA and protein of the factor were measured,and the expression of immunoregulatory factor was significantly decreased in the effective sequence of the transfected siFasL compared with the transfection-independent sequence group(P<0.05).(4)Two days after co-culture The proliferation and apoptosis of hPDLSCs in the two groups of transfection-insensitive sequences and transfected siFasL effective sequences were determined by flow cytometry.It was found that the effective sequence of transfected siFasL was higher than that of the transfected unrelated sequence group.The mortality rate was reduced(P<0.01).(5)After 48 hours of co-culture,the expression of ALP and Runx2 mRNA was detected by alizarin red staining,ALP staining and qRT-PCR.The expression of Runx2 was detected by Western blot.The effective sequence of transfected siFasL and transfection-independent sequence were found.The expression of osteogenic related factors was significantly lower(P<0.01 or P<0.05)Conclusions:(1)In this study,the enzymatic hydrolysis method and the limited dilution method were used in combination to obtain hPDLSCs,which were identified as one of the odontogenic sources of mesenchymal stem cells,so they have strong proliferative capacity and The potential for differentiation of bone and fat.(2)hPDLSCs have the ability to regulate immunity,mainly by secreting some soluble immunoregulators to regulate immune function and exert immunomodulatory effects.(3)siFasL can affect the immunomodulatory capacity of hPDLSCs,which is mainly regulated by inhibiting the secretion of soluble immunoregulatory factors by hPDLSCs.(4)siFasL is involved in the regulation of proliferation and apoptosis of hPDLSCs,which can promote the proliferation of hPDLSCs and inhibit its apoptosis.(5)siFasL is involved in the regulation of osteogenic differentiation of hPDLSCs,and low-level expression of FasL can reduce the osteogenic differentiation of hPDLSCs.