miR-429在低氧环境中对MC3T3-E1细胞成骨分化的影响  

Effect of miR-429 on Osteogenic Differentiation of MC3T3-E1 Cells in a Hypoxic Environment

作　　者：黄江[1] 焦旭峰 安帅[1] 王居勇[1] 曹光磊[1] 沈惠良[1] Huang Jiang;Jiao Xufeng;An Shuai;Wang Juyong;Cao Guanglei;Shen Huiliang(Department of Orthopaedics,Xuanwu Hospital,Capital Medical Univeristy,Beijing 100053,China)

机构地区：[1]首都医科大学宣武医院骨科,北京100053

出　　处：《实用骨科杂志》2022年第8期712-716,共5页Journal of Practical Orthopaedics

摘　　要：目的探讨在低氧环境中,miR-429对MC3T3-E1细胞成骨分化的影响。方法在体内、外用氯化钴(CoCl_(2))模拟低氧环境,采用免疫蛋白印迹(western blot,WB)检测低氧诱导因子(hypoxia inducible factor,HIF)-1α的表达验证低氧情况,通过实时荧光定量聚合酶链式反应(real time-quantitative polymerase chain reaction,RT-qPCR)观察骨折部位及细胞组织中miR-429的表达情况,同时检测MC3T3-E1细胞在低氧状态下的增殖与凋亡情况;通过慢病毒转染技术在MC3T3-E1细胞中过表达miR-429作为实验组,对照组为转染空载质粒细胞,采用碱性磷酸酶(alkaline phosphatase,ALP)活性检测、茜素红染色、WB等方法观察细胞中ALP活性、矿物质含量以及多种成骨标志物蛋白表达情况,对比两组MC3T3-E1细胞的成骨分化;在骨折模型局部注射过表达miR-429的慢病毒载体,通过Micro-CT及qRT-PCR观察骨折愈合情况。结果在低氧环境MC3T3-E1细胞中HIF-1α高表达的同时上调了miR-429的表达,对MC3T3-E1细胞转染miR-429过表达病毒后,细胞中ALP的活性、细胞基质矿化水平以及成骨标志物Runt相关转录因子2(Runt related transcription factor 2,RUNX2)和骨钙素(osteocalcin,OC)的水平均高于对照组;体内注射过表达miR-429的慢病毒试剂后,骨折Micro-CT愈合情况、骨组织中miR-429的表达均高于对照组。结论miR-429在低氧环境中呈高表达,在诱导MC3T3-E1细胞成骨分化中起到促进作用。Objective To investigate the effect of miR-429 on the osteogenic differentiation of MC3 T3-E1 cells in a hypoxic environment.Methods The hypoxia environment was simulated by Cobalt chloride(CoCl_(2))in vitro and in vivo,the expression of hypoxia-inducible factor-1α(HIF-1α)was detected by Western blotting to verify the hypoxia condition.The expression of miR-429 in cell tissues and the fracture site was observed by real time-quantitative polymerase chain reaction,(RT-qPCR).The proliferation and apoptosis of MC3 T3-E1 cells under hypoxia were detected.miR-429 was overexpressed in MC3 T3-E1 cells by lentiviral transfection technology as the experimental group and the control group was transfected with empty plasmid cells,and alkaline phosphatase(ALP)activity detection,alizarin red staining,western blotting and other methods were used to observe the ALP activity,mineral content and protein expression of various osteogenic markers in cells to verify the osteogenic differentiation of MC3 T3-E1 cells.The fracture model was injected locally with a lentiviral vector overexpressing miR-429,and the fracture healing was observed by Micro-CT and qRT-PCR.Results The high expression of HIF-1αin MC3 T3-E1 cells in a hypoxic environment up-regulated the expression of miR-429.After MC3 T3-E1 cells were transfected with miR-429 overexpressing virus,the activity of ALP in the cells,the mineralization of cell matrix and the levels of osteogenic markers(RUNX2 and OC)were higher than those in the control group.After injection of lentiviral reagents overexpressing miR-429 in vivo,the Micro-CT of fracture healing were better and the expression of miR-429 in bone tissue were higher than those in the control group.Conclusion MiR-429 is highly expressed in hypoxic environment and plays a role in inducing osteogenic differentiation of MC3 T3-E1 cells.

关 键 词：低氧诱导因子-1Α miR-429 成骨分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]