| BackgroundOsteonecrosis of the femoral head(ONFH)is one of the most common diseases leading to hip joint activity and functional limitations,which causes by ischemic changes in femoral head.Ischemic changes in femoral head will lead to Changes in the composition of bone marrow cells and the changes of the function and structure of the femoral head.The ONFH has a high disability rate.According to different etiology,Osteonecrosis of the femoral head(ONFH),which can be divided into traumatic ONFH and non-traumatic ONFH.While traumatic ONFH generally occurs after trauma,the causes of non-traumatic ONFH are complex,including abnormal circulating functions,alcohol addiction and steroid.In this one,the incidence of steroid ONFH is located in the first place caused by the incidence of steroid ONFH is widely used in the clinical work.Most of the patients often combined with other diseases,and bilateral femoral head involvement,which resulting in severe damage to the hip,thereby affecting the quality of life of patients.At present,the pathogenesis of steroid induced osteonecrosis of the femoral head is not fully understood,and there are no effective methods to prevent steroid induced osteonecrosis of the femoral head.The research on the effect of miRNA on the differentiation of stem cells has become more and more clear.However,the study of the relationship between miRNA and osteonecrosis of the femoral head has just begun.In the global report,there is no miRNA double targeting gene regulation to prevent steroid ONFH.Therefore,this study employ hormone induced rat model,using miRNA chip detection method to comprehensive analysis of hormone induced effects on miRNA expression,and select the miRNA-27a which is more closely related to femoral head necrosis.At the same time,according to the bioinformatics analysis,which found that the PPAR?gene and GREM1 gene,the BMP-2 gene,were all the target genes of miRNA-27a.I hope that this research has always been the regulation of the expression of PPAR?gamma and GREM1 expression of the target through the regulation of miRNA-27a,so as to effectively reduce the adipogenic differentiation induced by hormone,promote the osteogenic differentiation,and regulate the pathogenesis of femoral head necrosis.Therefore,the study of the application of miRNA double targeting gene regulation to prevent the occurrence of steroid ONFH is more innovative.ObjectiveBy regulating the expression of miRNA-27a go up,the down-regulation of PPAR?and GREM 1 expression was observed to investigate the effects of hormone on the adipogenic differentiation and osteogenic differentiation induced by BMSCs in rats.This study is divided into the following three parts:Part 1 miRNA analysis of specific expression in rat femoral headnecrosis induced by dexamethasoneMethods(1)Animal model making,grouping and HE staining:30 SD rats,female,were randomly divided into 2 groups,including model group and control group,the rats of model group were injected with dexamethasone sodium phosphate(10mg/kg)for 3times,24H for each time.At 4 weeks,remove the femoral head and paraffin embedded sections were then stained with HE.(2)The sifting of miRNA specific expression in ONFH:Two groups were randomly sacrificed in 3 rats,and the femoral heads were taken out and detected the miRNA microarray.(3)To verify the miRNA microarray results and detect the related gene expression:qRT-PCR was used to detect the expression of mi RNA-27a,PPAR?and GREM1 in the rat model of femoral head necrosis induced by glucocorticoid in rats.At the same time,the correlation between miRNA-27a and PPAR?gamma and GREM1 in the model of steroid induced osteonecrosis of the femoral head was analyzed.(4)SPSS 21.0 was used for the statistical analyses.Results(1)HE staining showed that the bone trabecula was thinner,sparser,smaller,and more commonly fractured and disordered in the Model group compared with the Control group.(2)The mi RNA microarray analysis identified there were 9 up-regulated miRNAs and 28 down-regulated miRNAs in the femoral heads from the Model group,which displayed at least a 3-fold change compared with the miRNA expression profiles in the Control group.(3)The result of qRT-PCR detection:the expression of miRNA-27a was significantly down regulated in the model group,miRNA-182 was significantly up regulated(P<0.05).(4)The expression levels of mRNA in PPAR?and GREM1 in the model group were significantly up regulated(P<0.05).(5)The result of correlation analysis:PPAR?and GREM1 were negatively correlated with miRNA-27a(R~2=0.717?R~2=0.662).Part2 the effect of up-regulation of miRNA-27a on the differentiationof bone marrow mesenchymal stem cells induced by hormone.Methods(1)Preparation of rat BMSCs:To isolate and culture rat BMSCs,flow cytometry was used to identify surface antigens CD29,CD34,CD45 and CD90.(2)The test of steroid induced BMSCs differentiation:The rat BMSCs induced by hormone were stained of fat by oil red"O",detected the intracellular triglyceride content,detected osteocalcin content in culture medium and alkaline phosphatase(ALP)activity.The rat BMSCs induced by hormone were detected the expression of PPAR?,GREM1,Runx2,BMP-2,C/EBP?was detected by qRT-PCR and Western blot.(3)The effect of miRNA-27a on steroid induced BMSCs differentiation:Up regulated the expression of mi RNA-27a by transfect miRNA-27a agomir in the rat BMSCs induced by hormone,there were detected the expression of miRNA-27a by qRT-PCR and detected the expression of PPAR?,GREM1,Runx2,BMP-2,C/EBP?was detected by qRT-PCR and Western blot.Up regulated the expression of miRNA-27a in the rat BMSCs induced by hormone,there were stained of fat by oil red"O"and stained by ALP staining.There were detected the intracellular triglyceride content,detected alkaline phosphatase(ALP)activity and osteocalcin content in culture medium.(4)SPSS 21.0 was used for the statistical analyses.Results(1)Flow cytometry to detect the surface markers showed that the third generation of rat BMSCs cultured have the expression of CD29 and CD90,the positive rate was 99.84%and 99.41%respectively;CD34 and CD45 were negative,the positive rate was 1.29%and 1.42%respectively.(2)The results of steroid induced BMSCs differentiationoil:The rat BMSCs induced by hormone for 14 days,oil red"O"staining in the rat BMSCs of model group showed that a large number of lipid droplets.The contents of triglyceride in the model group at 7 days and 14 days were significantly higher than the normal control group,the difference was statistically significant(P<0.05).The alkaline phosphatase and cell model group in culture medium osteocalcin content at 7 days and 14 days were significantly lower than the normal control group,the difference was statistically significant(P<0.05).The expression of PPAR?,GREM1 and C/EBP?were significantly higher in rats with hormone induced BMSCs at 7 and 14 days,and the difference was statistically significant(P<0.05).The expression of Runx2,Bmp-2and the expression of miRNA-27a were significantly lower than those in normal group,and the difference was statistically significant(P<0.05).(3)The results about the effect of miRNA-27a on steroid induced BMSCs differentiation:The expression of PPAR?,GREM1 and C/EBP were significantly higher in the model group and the unrelated sequence group than in the miRNA-27a group and the normal group,the difference was statistically significant(P<0.05).The expression of Runx2 and BMP-2 in the model group and the independent sequence group was significantly lower than that in the miRNA-27a group and the normal group,the difference was statistically significant(P<0.05).The rats BMSCs were stained with oil red O,A large number of lipid droplets were found in the BMSCs of rats in the model group and the unrelated control group,and the miRNA-27a group and the normal control group showed little.Alkaline phosphatase(ALP)staining showed that the BMSCs of the rats in the miRNA-27a group and the normal control group showed a large number of cells stained with blue/purple,and model group and the unrelated control group showed little.The contents of triglyceride in miRNA-27a group and normal control group of BMSCs rats were significantly lower than the model group and the unrelated control group,the difference was statistically significant(P<0.05).miRNA-27a group and normal control group rats BMSCs activity of alkaline phosphatase(ALP)and osteocalcin content in culture medium of cells were significantly higher than those in the model group and the unrelated control group,the difference was statistically significant(P<0.05).Part 3 Preliminary study on the mechanism of miRNA-27aregulating hormone induced differentiation of rat bonemarrow mesenchymal stem cellsMethods(1)Bioinformatics analysis and prediction of target genes:TargetScan and miRBase software for bioinformatics analysis and predict the target gene of miR-27a.(2)Identification of miRNA-27a target genes:Construction of pmirGLO-PPAR?and pmirGLO-GREM1 recombinant reporter gene vector,transfection of mi RNA-27a agomir and mi RNA-27a scramble in BMSCs of the rats,and Confirm the target gene of miR-27a by the dual luciferase reporter assay.(3)The comparison of Up regulated the miRNA-27a and silence the target genes on steroid induced BMSCs differentiation:experiment were divided into 5 groups,Blank group(rats BMSCs);Model group(BMSCs+dexamethasone);mi RNA-27a group(miRNA-27a transfected into BMSCs+dexamethasone);Si-PPAR?group(the si-PPAR?transfected into BMSCs+dexamethasone);si-GREM1 group(the si-GREM1 transfected into BMSCs+dexamethasone).After 24 hours of transfection,the qRT-PCR method was used to detect the expression of miRNA-27a.The rat BMSCs culture for 14 days,the rat BMSCs were detected the expression of PPAR?,GREM1,Runx2,BMP-2,C/EBP?was detected by qRT-PCR and Western blot.The rat BMSCs culture for 14 days,Each group of the rat BMSCs were stained of fat by oil red"O"and stained by ALP staining.Each group of the rat BMSCs were detected the intracellular triglyceride content;alkaline phosphatase(ALP)activity and osteocalcin content in culture medium.(4)SPSS 21.0 was used for the statistical analyses.Results(1)Using TargetScan and miRBase for bioinformatics analysis and prediction,the results show that:PPAR?and GREM1 are the targets of miRNA-27a.(2)Dual luciferase reporter assay showed that the miR-27a can reach the negative regulation of UTR expression by acting on the 3'region of PPAR?and GREM1.(3)24 hours after transfection,the expression level of miRNA-27a in BMSCs of rats was significantly increased,and significantly higher than the other 4 groups,it was explained that the success of miRNA-27a,the difference was statistically significant(P<0.05).(4)The rat BMSCs culture for 14 days,the expression of PPAR?in model group and si-GREM1 group were significantly higher than those in the normal group,si-PPAR?group and miRNA-27a group,the difference was statistically significant(P<0.05).The expression of GREM1 in the model group and si-PPAR?group was significantly higher than that of normal group,si-GREM1 group and mi RNA-27a group,the difference was statistically significant(P<0.05).The expression of Runx2in the control group,si-GREM1 group and miRNA-27a group were significantly higher than si-PPAR?group and model group,the difference was statistically significant(P<0.05).The expression of BMP-2 in the model group and the si-PPAR?gamma group was significantly lower than the normal group,si-GREM1 group and miRNA-27a group,the difference was statistically significant(P<0.05).The expression of C/EBP?in the model group and si-GREM1 group was significantly higher than that in normal group,si-PPAR?group and miRNA-27a group,with statistically significant difference(P<0.05).(5)The rat BMSCs culture for 14 days,the rats BMSCs were stained with oil red O,there were a lot of lipid droplets in the model of rat BMSCs in model group and si-GREM1 group,but normal group,si-PPAR?group and miRNA-27a group showed little.Alkaline phosphatase(ALP)staining showed that the BMSCs of the rats in normal group and si-GREM1 group and miRNA-27a group showed that a lot of cytoplasmic staining for blue/purple,but model group and si-PPAR?group showed little.(6)The rat BMSCs culture for 14 days,in normal group,si-PPAR?group and miRNA-27a group,the triglyceride content of BMSCs was significantly lower than that of the model group and si-GREM1 group,the difference was statistically significant(P<0.05).(7)The rat BMSCs culture for 14 days,in the normal group,si-GREM1 group and mi RNA-27a group the alkaline phosphatase(ALP)activity and osteocalcin content of cell culture medium were significantly higher than those in the model group and the si-PPAR?group,the difference was statistically significant(P<0.05).Conclusion(1)In the rat model of femoral head necrosis induced by hormone,the expression of miRNA-27a is significantly down regulated,the expression of PPAR?,GREM1 is obviously up regulated.There was a negative correlation between the expression of miRNA-27a and PPAR?and GREM1.(2)Up regulation of miRNA-27a expression in rat BMSCs can effectively inhibit the adipogenic differentiation of rat BMSCs induced by hormone,and promote the osteogenic differentiation of rat BMSCs.(3)It has been proved that miRNA-27a can be combined with the 3'UTR of PPAR?and GREM1,and negatively regulate the expression of them,which can play a part in the differentiation of rat BMSCs. |
PDF Full Text Request