摘要
目的探讨microRNA-494(miR-494)通过Toll样受体-4(TLR-4)通路抑制成骨细胞分化及基质矿化的分子机制。方法选用小鼠成骨细胞株MC3T3-E1作为体外模型,采用miR-494 mimic转染小鼠成骨细胞株MC3T3-E1,采用qRT-PCR法测定miR-494在小鼠成骨细胞株MC3T3-E1转染前后的表达情况;通过MTT法检测转染前后小鼠成骨细胞株MC3T3-E1的增殖情况;通过测定碱性磷酸酶(alkaline phosphatase,ALP)活性,研究小鼠成骨细胞株MC3T3-E1的分化情况;通过骨钙素(osteocalcin,OC)定量检测分析和Von Kossa钙化染色检测小鼠成骨细胞株MC3T3-E1的基质矿化情况;通过Western blot等检测转染前后TLR-4蛋白的表达差异。结果 qRT-PCR测定结果显示,与阴性对照组相比,小鼠成骨细胞株MC3T3-E1转染后的miR-494的mRNA的表达量升高(P<0.05),说明miR-494 mimic转染小鼠成骨细胞株MC3T3-E1,成功使miR-494过表达。与阴性对照组相比,经过miR-494 mimic转染后,小鼠成骨细胞株MC3T3-E1的增殖能力受到抑制(P<0.05)。转染后的小鼠成骨细胞株MC3T3-E1中ALP的活性降低(P<0.05)。同时,转染后的小鼠成骨细胞株MC3T3-E1中OC的活性显著降低(P<0.05),矿化结节的数目、减少(P<0.05)。Western blot结果显示,与阴性对照组相比,miR-494 mimic转染组细胞中的TLR-4蛋白表达量显著升高(P<0.05)。结论 miR-494能够通过TLR-4通路抑制小鼠成骨细胞株MC3T3-E1的增殖活力,降低细胞中ALP、OC的活力,减少成骨细胞中的矿化结节数,抑制成骨细胞分化及基质矿化。
Objective In this study,the molecular mechanism by which microRNA-494 inhibits osteoblast differentiation and matrix mineralization through the TLR-4 pathway was examined.Methods The mouse osteoblast strain MC3T3-E1 was selected as an in vitro model,which was transfected with miR-494 mimic.The expression of miR-494 before and after this transfection was determined by qRT-PCR.The proliferation of MC3T3-E1 before and after transfection was also detected by MTT assay,while the differentiation was studied by measuring ALP activity.In addition,matrix mineralization of this cell line was determined by quantitative detection of osteocalcin(OC)and Von Kossa calcification staining.Finally,the difference of TLR-4 protein expression before and after transfection was determined by Western blotting.Results Compared with that in the negative control group,the mRNA expression of miR-494 after transfection of the mouse osteoblast cell line MC3T3-E1 was significantly increased(P<0.05).The result showed that miR-494 mimic was transfected into this cell line and successfully led to the overexpression of miR-494.Compared with the negative control group,after transfection with miR-494 mimic,the proliferative capacity of mouse MC3T3-E1 osteoblasts was inhibited(P<0.05).ALP activity in MC3T3-E1 was also significantly decreased after transfection(P<0.05),indicating that the differentiation ability of MC3T3-E1 cells was inhibited.At the same time,the activity of OC in MC3T3-E1 was significantly decreased after transfection(P<0.05)and the number of mineralized nodules was also significantly decreased(P<0.05),which indicated that the overexpression of miR-494 inhibited the mineralization of matrix in these cells.Western blot result showed that TLR-4 protein expression was significantly increased in cells transfected with miR-494 mimic,compared with that in the negative control group(P<0.05).Conclusions miR-494 could inhibit the proliferative activity of the mouse osteoblast line MC3T3-E1 through the TLR-4 pathway,reduce the activity of ALP and OC in cells,decrease the number of mineralized nodules in osteoblasts,and inhibit osteoblast differentiation and matrix mineralization.
作者
匡嘉兵
徐昊
张克良
沈波
许闫严
KUANG Jiabing;XU Hao;ZHANG Keliang;SHEN bo;XU Yanyan(Department of orthopedics,Wuhan Pu AI hospital(Pu Yu Hospital Affiliated to Huazhong University of Science and Technology),Wuhan 430030,China)
出处
《中国比较医学杂志》
CAS
北大核心
2018年第10期55-60,共6页
Chinese Journal of Comparative Medicine
作者简介
匡嘉兵,(1983—),男,学士,专业:成骨分化研究。E-mail:9592662@qq.com;通信作者:徐昊,(1969—),男,学士,专业:骨科研究。E-mail:jmxfxf@163.com
