| Part One Correlation analysis between MGAT5 and postmenopausal osteoporosisObjective: To study the correlation between MGAT5 and bone mineral density(BMD)by detecting the expression level of MGAT5 and bone mineral density in clinical postmenopausal osteoporosis patients and age-matched healthy patients.Methods:1.A total of 60 postmenopausal women were enrolled and divided into postmenopausal osteoporosis group(PMOP group)and postmenopausal nonosteoporosis group(Control group),with 30 women in each group.The general characteristics of the clinical trial subjects were collected,such as age,age at menopause,body mass index(BMI),calcium,phosphorus,and hemoglobin,total cholesterol,etc.2.Bone mineral density was measured by dual-energy X-ray absorbometry(DXA).3.The expression of MGAT5 m RNA in serum was detected by Real time PCR.Results: We found that BMD of left femoral neck was positively correlated with the expression level of MGAT5,age at menopause and the content of hemoglobin,but negatively correlated with alkaline phosphatase in patients with osteoporosis.After revising for age at menopause,hemoglobin content,and alkaline phosphatase,the expression level of MGAT5 was associated with an decreased risk of osteoporosis.Conclusions: The expression level of MGAT5 has been found to have a protective effect on osteoporosis through clinical studies.MGAT5 expression may regulate the occurrence of osteoporosis by affecting the balance between osteogenesis and osteoclast.Part Two Effects of ovariectomy on MGAT5 and bone metabolism in miceObjective: The animal model of postmenopausal osteoporosis was established by bilateral ovariectomy in female C57BL/6J mice.Based on the animal model,the degeneration of bone microstructure and MGAT5 expression level in postmenopausal osteoporosis mice were investigated.Methods:A total of 12 healthy 7-week-old female C57BL/6J mice were fed adaptively for one week before establishing the model.Mice were rando-mly divided into two groups: Control group and OP group.1.Bone microstructure parameters were analyzed by Mirco-CT to observe the degeneration of bone microstructures in ovariectomized mice.2.Western blot was used to detect the protein expression of MGAT5 and osteogenic markers RUNX2,OCN and OSX in vertebrae and femur.3.The m RNA expression level of MGAT5 and osteogenic markers RUNX2,OCN and OSX in vertebrae and femur were detected by Real time PCR.4.Serum ALP activity was measured with the ALP assay kit.Results:1.At 6 weeks after operation,Micro-CT images showed that the trabecular bone of the femur in the OP group was significantly sparse compared with the Control group,and the number of trabecular bone was significantly reduced.BMD(bone mineral density),BV/TV(bone volume fraction)and Tb.N(trabecular bone number)were significantly decreased in the OP group compared with the Control group(P<0.05).2.At 6 weeks after operation,the protein expression levels of MGAT5 in vertebrae and femur in OP group were significantly lower than those in Control group(P<0.05);At 6 weeks after operation,the protein expression levels of RUNX2,OCN and OSX in vertebrae and femur in OP group were significantly lower than those in Control group(P<0.05).3.At 6 weeks after operation,the m RNA expression levels of MGAT5 in the vertebrae and femur of the OP group were significantly lower than those of the Control group(P<0.05).At 6 weeks after operation,the m RNA expression levels of RUNX2,OCN and OSX in the vertebrae and femur of the OP group were significantly lower than those of the Control group(P<0.05).4.At 6 weeks after operation,the serum alkaline phosphatase(ALP)activity was increased in the OP group compared with the Control group(P<0.05).Conclusions: The animal model of postmenopausal osteoporosis can be successfully established by ovariectomy,and the animal model is consistent with the characteristics of human postmenopausal osteoporosis.The expression level of MGAT5 had the same trend with the development of PMOP,which may be involved in the development of OP and related to the osteogenic differentiation of BMSCs.Part Three Effect and mechanism of MGAT5 on osteogenic differen-tiation of bone marrow mesenchymal stem cellsObjective: To investigate the effect of MGAT5 on the osteogenic differentiation of mouse bone marrow mesenchymal stem cells and its mechanism.Methods: Bone marrow derived mesenchymal stem cells of C57BL/6J female mice were isolated and cultured in vitro,and the BMSCs at passage 3were obtained.1.BMSCs were identified by their immunophenotype and multipotent differentiation ability.2.BMSCs were incubated with osteogenic induction medium,and the osteogenic differentiation ability of BMSCs was observed by ALP staining at7 days.At 14 days,Alizarin red S staining was used to observe osteogenic differentiation and the formation of bone mineralized nodules.3.The m RNA and protein expression levels of MGAT5 were detected by Real time PCR and Western blot,and the m RNA expression levels of RUNX2,OCN and OSX were detected by Real time PCR on day 0,7 and 14 of incubation in normal medium and osteogenic medium,respectively.4.The LV-sh MGAT5 vector virus was constructed and transfected into BMSCs,and then the BMSCs with MGAT5 knockdown were obtained.After detecting the effective knockdown of MGAT5,the BMSCS were inducted to osteogenic differentiation for 7 days.The expression levels of MGAT5,RUNX2,OCN and OSX in the cells were detected by Western blot and Real time PCR.ALP activity in the cell supernatant was detected by ALP assay kit,and osteogenic activity was observed by ALP staining.5.After 14 days of induction,Alizarin red S staining was performed to observe osteogenic differentiation.After 14 days of adipogenic differentiation induction,oil Red O staining was performed to observe adipogenic differentiation.6.BMSCs with MGAT5 knockdown were incubated with osteogenic medium for 7 days,The nuclear β-catenin protein expression and the protein expressions of BMP2,SMAD1 and p-SMAD1(Ser463/465),TGFβ1,SMAD2,p-SMAD2(Ser465/467),SMAD3 and p-SMAD3(Ser423/425)in the cells were detected by Western blotting analysis.The m RNA expression level of c-myc and axis inhibition protein 2(Axin2)was detected by Real-time PCR.Results:1.When BMSCs were incubated in vitro,the expression levels of MGAT5,ALP,RUNX2,OCN and OSX were significantly increased with the induction of osteogenic differentiation(P<0.0.5).2.The expression levels of MGAT5,ALP,RUNX2,OCN and OSX were significantly decreased after 7 days of osteogenic differentiation in BMSCs with effective knockdown of MGAT5(P<0.0.5).3.The osteogenic differentiation ability of BMSCs with effective knockdown of MGAT5 was weakened and the adipogenic differentiation ability was intensified.4.MGAT5 may participate in the osteogenic differentiation of BMSCs by regulating Wnt/β-catenin signaling pathway and BMP/TGF-β signaling pathway.Conclusions: MGAT5 was involved in the osteogenic differentiation of BMSCs,and its loss aggravated the adipogenic differentiation of BMSCs.MGAT5 may regulate the osteogenic differentiation of BMSCs through Wnt/β-catenin signaling pathway and BMP/TGF-β signaling pathway,and its loss may be related to the development of OP. |
PDF Full Text Request