Effect Of IRS-1 On Adipogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective: With the development of population aging,the incidence of osteoporosis is increasing year by year,which brings serious burden to individual,family and society.It has been found that the occurrence of osteoporosis is related to the imbalance of bone remodeling mechanism,and the directional differentiation of bone marrow mesenchymal stem cells plays an important role in this process.Transcriptional coactivator with pdz-binding motif(TAZ)plays an important role in maintaining the balance of adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells.In recent years,it has been found that insulin receptor substrate-1(IRS-1)can affect the adipogenic differentiation of bone marrow mesenchymal stem cells,but its specific inhibitory or promotive effects are still controversial.And the specific mechanism of its action is still not very clear.To explore whether IRS-1 could affect the adipogenic differentiation of BMSCs and whether this role is played by influencing the relative expression of TAZ,We used plasmid transfection technique to silence IRS-1 gene and tested the relative expression of PPARγ as well as TAZ by reverse transcription quantitative PCR.Methods:1.SD rat bone marrow mesenchymal stem cells were isolated by whole bone marrow adherent method.Then it was cultured in primary culture and its purity was determined.Using adipogenic induction(10mg/ml insulin,100 mM indometacin,1mM dexamethasone,500 mM IBMX)to induce the adipogenic differentiation of BMSCs and assess its ability of adipogenesis.2.Irs-1 gene silencing bone marrow mesenchymal stem cells(BMSCs)model was constructed by transfection of Si-IRS-1 plasmid into bmscs by plasmid transfection technique.3.We tested the relative expression of PPARγ by reverse transcription quantitative PCR(RT-qPCR).4.We tested the relative expression of TAZ by reverse transcription quantitative PCR(RT-qPCR).5.We analyzed the experimental data by SPSS 21.0.Results:1.The results of bmscs surface antigen identification obtained by whole bone marrow adherent method showed that The positive rate of CD29 expression was 99.9% and the positive rate of CD90 was 93.5%,the positive rate of CD34 was 0.7%and the positive rate of CD45 was 1.8%.After induced by lipogenic inducer for 14 days,the changes of bmscs lipid formation were obvious.The results of oil red O test showed that the OD value of adipogenesis group was significantly higher than that of control group.The difference was statistically significant(P < 0.05).The level of PPAR?increased significantly at the early stage of adipogenic induction,and increased with time.The difference was statistically significant(P<0.05).2.Compared with the control group,the expression of PPARγ in the SiIRS-1 group was significantly higher than that in the con group,and the difference was statistically significant(P<0.05).3.Compared with the control group,the expression of TAZ in the Si-IRS-1group was significantly higher than that in the con group,and the difference was statistically significant(P<0.05).Conclusion:1.Bone marrow mesenchymal stem cells obtained by whole bone marrow adherent method are of high purity.2.The level of PPARγ mRNA increased significantly at the early stage of adipogenic differentiation,and increased with adipogenesis time during the 14 days of the experiment.3.Irs-1 inhibits adipogenic differentiation of bone marrow mesenchymal stem cells by increasing the level of taz mran in cells.