| BackgroundOsteonecrosis of the femoral head(ONFH)is a complex disease caused by genetic susceptibility factors and environmental factors.The diseased bone marrow cavity is often filled with a large amount of adipose tissue,so the disorder of lipid metabolism is recognized as the core pathogenesis of ONFH.With the increasing incidence of ONFH and its increasingly prominent threat to human health,the molecular pathogenesis of ONFH has increasingly become a hot spot in the field of orthopaedics.Animal experiments and clinical studies have confirmed that a high-fat environment can inhibit the differentiation of osteoblasts.The increase of adipocytes in bone marrow after hyperlipidemia will oppress the microvessels of the femoral head and lead to ONFH.It has been speculated that the abnormal transdifferentiation of adipogenesis and osteogenesis may play a key role in the occurrence and development of ONFH,but the molecular mechanism of adipocyte accumulation has not been clear.In recent years,studies on the differentiation microenvironment of bone marrow mesenchymal stem cells(BMSCs)have found that the key transcription factor PPARγ(peroxisome proliferator-activated receptor γ)plays a key role in regulating adipogenic and osteogenic differentiation.As the main regulator of adipogenesis and differentiation,PPARγ is widely used as a molecular marker of adipogenesis and differentiation in the molecular cascade of adipocyte and BMSCs differentiation.Our team recently found that the rs2920502 mutation(CC type)of the PPARγ gene promoter was significantly associated with the risk of ONFH in the Chinese Han population by mass spectrometry,and the analysis showed that the contents of serum triglyceride(TG),low-density lipoprotein(LDL-c)and LDL-c/HDL-c ratio were significantly increased and the content of high-density lipoprotein(HDL-c)was significantly decreased in patients with ONFH.At the same time,the CC genotype of this promoter mutation was significantly related to the level of serum TG in patients with ONFH.This study first confirmed that mutations in the PPARγ gene promoter,which is the main regulator of adipocyte formation,are significantly associated with the risk of ONFH and its lipid metabolic disorders.The progress of the research on the molecular mechanism of ONFH is to confirm that several susceptible genes are associated with the risk of ONFH,but due to the lack of functional verification of mutant genes,it is difficult to confirm their role in the occurrence of ONFH.Through the unique regulatory effect of PPARγ on osteogenic and adipogenic differentiation and our new finding that PPARγ gene promoter mutation is involved in the risk of ONFH,PPARγ may play a key role in initiating and aggravating the accumulation of adipocytes in the femoral head of ONFH.To further explain the molecular mechanism of PPARγ promoter mutation in ONFH,an ONFH case-control system was established.BMSCs were isolated and cultured in vitro and genotyped by PPARγ promoter rs2920502 locus.The effects of different PPARγ promoter rs2920502 loci on adipogenic osteogenic transdifferentiation of BMSCs were systematically studied.The effects of different rs2920502 loci of PPARγ promoter on the expression of BMSCs PPARγ transcription and translation and the effects of PPARγ promoter mutation on the key molecular markers involved in adipogenic osteoblast transdifferentiation were studied,and the effect of PPARγ promoter mutation on its activity was verified by the luciferase reporter gene.This study analyzed the relationship between PPARγ promoter mutation and ONFH adipocyte accumulation and clarified the molecular mechanism of ONFH adipocyte accumulation caused by PPARγ promoter mutation.The purpose of this study is to provide a scientific basis and potential molecular targets for the establishment of molecular prevention and treatment of ONFH and to promote the connection between molecular etiology research and early treatment of ONFH.Method1.Bone marrow blood 10 ml was collected from 54 patients with ONFH and 32 patients with femoral neck fracture in joint surgery,and BMSCs were isolated by gradient centrifugation and cultured in low glucose DMEM medium.The morphology and growth of cells were observed by phase-contrast microscope.2.The cell surface antigen and cell cycle of BMSCs were detected by flow cytometry,and the ability of cell proliferation was detected by CCK-8 method.3.PPARγ rs2920502 locus was genotyped by Sanger sequencing in 54 cases of the ONFH group and 32 cases of control group BMSCs.4.Osteogenic and adipogenic differentiation induction kits were used to induce BMSCs to differentiate into adipocytes and osteoblasts.The osteogenic and adipogenic ability of BMSCs was analyzed by alizarin red staining(ARS),oil red O staining and quantitative extraction5.qRT-PCR and Western blot methods were used to detect the expression of PPARγ and related genes of osteogenesis and adipogenesis in the process of adipogenic osteogenesis of BMSCs.6.The promoter fragment of PPAγ gene was cloned from the DNA genome of BMSCs cells with rs2920502 GG and CC types by PCR technique,and the promoter fragment was recombined into the luciferase reporter gene lentiviral expression vector to construct a lentiviral vector that can detect the activity of rs2920502 promoters to regulate gene expression in BMSCs.7.The lentivirus was packaged and prepared by 293 FT cells,and the lentivirus titer was calculated by flow cytometry.The lentivirus was packaged to infect BMSCs cells,and the virus infection was observed by a fluorescence microscope.8.The promoter activity of different PPARγ rs2920502 genotypes was evaluated by luciferase activity assay.Results1.BMSCs were successfully isolated and cultured from bone marrow blood of proximal femoral shaft in 54 patients with ONFH and 32 patients with a femoral neck fracture.The cultured BMSCs accorded with the morphological characteristics of mesenchymal stem cells.The expression of CD73,CD90 and CD105 antigens on the cell surface was positive,while the expression of CD34 and CD45 antigens were negative.There was no significant difference in the cell cycle of BMSCs isolated and cultured between ONFH and the control group.The proliferation ability of BMSCs in the ONFH group was slightly lower than that in the control group,but there was no significant difference between the two groups.2.Oil red O and ARS and their quantitative analysis showed that the adipogenic ability of ONFH was significantly stronger than that of the control group(P<0.05),and the osteogenic ability of ONFH was significantly weaker than that of the control Xgroup(P<0.05).The expression of BMSCs adipogenic differentiation genes detected by qRT-PCR showed that the expressions of BMSCs adipogenic markers PPARγ,retinoid X receptor alpha(RXRα),CCAAT enhancer-binding protein alpha(CEBPα),lipoprotein lipase(LPL)and Adipsin in ONFH group were significantly higher than those in the control group(P<0.05).The expression of BMSCs osteogenesis-related markers including Runt related transcription factor2(RUNX2),alkaline phosphatase(ALP),bone morphogenetic protein 2(BMP2)and osteopontin(OPN)in ONFH group was significantly lower than those in the control group(P<0.05)This part of the study describes the characteristics and key molecular targets of ONFH BMSCs adipogenic osteogenic transdifferentiation disorder.3.The genotyping of the ONFH case-control system BMSCs PPARγ promoter rs2920502 was completed for the first time.Among them,there were 32 cases of GG type,19 cases of GC type and 3 cases of CC in ONFH group,and 16 cases of GG type,15 cases of GC type and 1 case of CC type in control group according to rs2920502 classification.The results of genotyping were consistent with the results of genomic typing of peripheral blood mononuclear cells in the case-control system of ONFH,which laid a foundation for further elucidating the effect of PPARγ promoter mutation on adipogenic osteogenic transdifferentiation.4.The results of adipogenic osteogenic differentiation ability of different genotypes of BMSCs showed that the adipogenic ability of BMSCs PPARγ promoter rs2920502 CC type in ONFH group and control group was significantly higher than that of GG type,but the osteogenic ability was significantly lower than that of GG type.It was the first to explain the evidence that PPARγ promoter mutation affected the adipogenic osteogenic differentiation ability of BMSCs.5.The results of the study on the effect of PPARγ promoter mutation on its gene expression showed that on the 7th day after adipogenic induction,the expression of PPARγ of BMSCs CC type in both groups was significantly higher than that of GG type and GC type(P<0.05)On the 14 th day after adipogenic induction,the expression of PPARγ of CC genotype in the ONFH group was significantly higher than that of GG and GC genotypes,and the expression of PPARγ of CC genotype in the control group was also significantly higher than that of GG genotype(P<0.05).The analysis of PPARγ expression of different BMSCs genotypes between ONFH group and control group showed that on the 7th day of adipogenesis,the expression of PPARγ of GC type and CC type in ONFH group was significantly higher than that of GC type and CC type of control group.On the 14 th day of adipogenesis,the expression of PPARγ of GG type,GC type and CC type in the ONFH group was significantly higher than that of GG type,GC type and CC type of control group(p<0.05).The experimental evidence that the PPARγ promoter mutation affects its gene expression was first proposed.6.The results of the study on the key molecular markers related to PPARγ promoter mutation showed that after adipogenic induction,the expression of BMSCs CC adipogenic markers RXR α,CEBP α and LPL in ONFH group and control group was significantly higher than that in GG type(P<0.05).After osteogenesis induction,the expression of RUNX2 CC type osteogenic markers of BMSCs in the ONFH group and the control group was significantly lower than that of GG type(P<0.05),and The expression level of CC type osteogenic marker Osterix of BMSCs in ONFH group was also significantly lower than that of GG type.The expression levels of RXRα,CEBPα,LPL and Adipsin of GG,GC and CC types in the ONFH group were significantly higher than those of GG,GC and CC types in the control group(P<0.05).And the expression levels of GG,GC and CC osteogenic markers RUNX2,BMP2,OPN,ALP and Osterix in the ONFH group were significantly lower than those of the control group.This study is the first to explain the key molecular targets of ONFH BMSCs with adipogenic and osteogenic transdifferentiation caused by mutations in the PPARγ promoter.7.PPARγ promoter rs2920502 lentiviral expression vectors LV-pPPARγ-GG and LV-pPPARγ-CC were successfully constructed.The sequence and insertion direction was confirmed by restriction endonuclease digestion and gene sequencing.The lentivirus vector was successfully packaged in 293 FT cells with a lentivirus titer of 3.49x108 TU/mL.The results of the luciferase activity assay showed that there was no significant difference in luciferase activity between the cells transfected with LV-pPPARγ-GG and LV-pPPARγ-CC lentiviral particles 72 hours after lentiviral vector infection.On the 7th,10 th and 14 th days of adipogenic induction,the luciferase activity of LV-pPPARγ-CC was significantly higher than that of LV-pPPARγ-GG lentiviral vector group(P<0.05).It was the first to explain the enhancement of PPARγ promoter activity caused by PPARγ promoter rs2920502 mutation.This study provides an innovative idea for the molecular mechanism of the disorder of adipogenic osteogenic transdifferentiation in ONFH and also provides a potential molecular target for the prevention and treatment of ONFH at the molecular level.The results of this study suggest that the normal function of the PPARγ promoter maintains the role of PPARγ as the main regulator of adipogenic differentiation.It dominates the microenvironment of BMSCs differentiation and the function of its main target molecules differentiate towards osteogenesis,while adipogenic differentiation is inhibited.However,the mutation of PPARγ gene promoter rs2920502(CC type)leads to the increase of promoter activity and the enhancement of PPARγ gene expression and dysfunction.The promoter mutation causes the disorder of osteogenic and adipogenic differentiation function of PPARγ-dominated BMSCs differentiation microenvironment,changes the BMSCs differentiation microenvironment,and weakens osteogenic differentiation and enhances adipogenic differentiation.Affected by environmental factors,adipose tissue in the bone marrow cavity of the femoral head gradually accumulates,which leads to the occurrence and development of ONFH.The results of this study were the first to analyze the disorder of ONFH BMSCs adipogenesis and osteogenic transdifferentiation caused by PPARγ promoter mutation.And promoter mutation can cause and aggravate adipocyte accumulation of ONFH through its key molecular targets.This study analyzed the internal relationship between PPARγ promoter mutation,abnormal regulatory molecular spectrum and ONFH adipocyte accumulation,and further expounded the molecular mechanism of ONFH adipocyte accumulation caused by PPARγ promoter mutation.It provides the basis and key molecular targets for the establishment of molecular level prevention and control strategies for ONFH and laid a foundation for further screening and early intervention of people susceptible to ONFH. |
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