摘要
目的 探究miR-494在骨质疏松大鼠中的表达及其调控机制。方法 选取30只8周龄雌性Sprague-Dawley大鼠,构建骨质疏松大鼠模型。双能X线吸收仪测定骨密度。ELISA检测大鼠血清中骨钙素(BGP)和总碱性磷酸酶(TALP)含量。分类培养大鼠骨髓间充质干细胞(BMSCs)。qRT-PCR检测miR-494和TLR4 mRNA的表达。Western blot检测TLR4蛋白的表达。双荧光素酶报告基因验证miR-494与TLR-4的靶向关系。CCK8实验检测各组BMSCs的增殖能力。茜素红S染色检测各组BMSCs成骨分化情况。油红O染色检测各组BMSCs成脂分化情况。结果 和NC组相比,OP组大鼠骨密度、BGP含量、细胞增殖能力、矿化结节数均显著降低,TALP含量、miR-494和TLR4的表达、脂肪细胞数均显著升高(P<0.05)。和OP组相比,OP+miR-494 inhibitor组大鼠骨密度、BGP含量、细胞增殖能力、矿化结节数均显著升高,TALP含量、miR-494和TLR4的表达、脂肪细胞数均显著降低(P<0.05)。和OP+miR-494 inhibitor组相比,OP+miR-494 inhibitor+TLR-4 vector组大鼠骨密度、BGP含量、细胞增殖能力、矿化结节数均显著降低,TALP含量、miR-494和TLR4的表达、脂肪细胞数均显著升高;OP+miR-494 inhibitor+sh TLR-4组大鼠骨密度、BGP含量、细胞增殖能力、矿化结节数均显著升高,TALP含量、miR-494和TLR4的表达、脂肪细胞数均显著降低(P<0.05)。结论 miR-494通过促进TLR-4通路的表达,加快大鼠骨质疏松的进展,为骨质疏松的发病机制提供新的理论依据。
Objective To explore the mechanism of miR-494 on the pathogenesis of osteoporosis in rats by targeting TLR-4 pathway.Method Thirty 8-week-old female Sprague Dawley rats were selected to construct the rat model of osteoporosis.Bone mineral density was measured by dual energy X-ray absorptiometry.The contents of osteocalcin(BGP) and total alkaline phosphatase(TALP) in rat serum were detected by ELISA.Rat bone marrow mesenchymal stem cells(BMSCs) were cultured.qRT-PCR was used to detect the expression of mir-494 and TLR4 mRNA.The expression of TLR4 protein was detected by Western blot.Double luciferase reporter gene verified the targeting relationship between miR-494 and TLR-4.The proliferation ability of BMSCs in each group was detected by CCK8 experiment.Alizarin red S staining was used to detect the osteogenic differentiation of BMSCs in each group.The adipogenic differentiation of BMSCs in each group was detected by oil red O staining.Result Compared with NC group,bone mineral density,BGP content,cell proliferation ability and the number of mineralized nodules in OP group were significantly decreased,while the content of TALP,the expression of miR-494 and TLR4 and the number of adipocytes were significantly increased(P<0.05).Compared with OP group,the bone mineral density,BGP content,cell proliferation ability and the number of mineralized nodules in OP + miR-494 inhibitor group were significantly increased,while the content of TALP,the expression of miR-494 and TLR4 and the number of adipocytes were significantly decreased(P<0.05).Compared with OP + miR-494 inhibitor group,the bone mineral density,BGP content,cell proliferation ability and the number of mineralized nodules in OP + miR-494 inhibitor + TLR-4 vector group were significantly decreased,while the content of TALP,the expression of miR-494 and TLR4 and the number of adipocytes were significantly increased;In OP + miR-494 inhibitor + sh TLR-4 group,bone mineral density,BGP content,cell proliferation ability and the number of mineralized nodules increased significantly,while the content of TALP,the expression of mir-494 and TLR4 and the number of adipocytes decreased significantly(P<0.05).Conclusion MiR-494 can accelerate the progress of osteoporosis in rats by promoting the expression of TLR-4 pathway.To provide a new theoretical basis for the pathogenesis of osteoporosis.
作者
匡嘉兵
郭松
雷建平
程煜方
KUANG Jiabing;GUO Song;LEI Jianping;CHENG Yufang(Department of Orthopedics,Wuhan Fourth Hospital,Wuhan 430060)
出处
《实验动物科学》
2023年第6期19-25,共7页
Laboratory Animal Science
基金
湖北省武汉市卫生健康科研基金(WX21Q63)。
作者简介
匡嘉兵(1983-),男,硕士研究生,主治医师,研究方向:主要从事西医创伤骨科的研究.E-mail:cf2018zx@163.com;通信作者:程煜方(1970-),男,本科,副主任医师,研究方向:西医创伤骨科的研究.E-mail:tr349ex@163.com。
