Dlx2在MC3T3-E1细胞成骨分化过程中的作用

目的:探讨Dlx2(distal-less homeobox 2)基因过表达对体外培养的小鼠前成骨细胞系MC3T3-E1 向成骨方向分化的影响.方法:构建Dlx2过表达的反转录病毒载体,测序验证.体外病毒转染MC3T3-E1后,以嘌呤霉素行抗性筛选稳定细胞株,以RT-PCR和Western印迹检测转染后细胞中Dlx2的表达.应用实时定量PCR(RT-PCR)检测Dlx2过表达对部分成骨相关基因(ALP、OCN、Runx2、Msx2)表达的影响.以碱性磷酸酶(ALP)活性测定和茜素红染色法检测Dlx2过表达对MC3T3-E1细胞成骨分化的影响.采用SAS 6.04软件包对数据进行随机设计的方差分析.结果:本实验成功构建Dlx2过表达的反转录病毒载体,测序正确.体外转染后筛选出Dlx2稳定高表达细胞株MC3T3-E1-Dlx2,其mRNA和蛋白表达量显著高于对照组.在成骨诱导过程中,MC3T3-E1-Dlx2细胞ALP值在第4、7、14d均高于对照组细胞,茜素红染色显示实验组较对照组和空白组染色深.Dlx2过表达在成骨诱导早期能显著促进ALP和Msx2的表达(P<0.05),晚期则上调OCN表达(P<0.05),而Runx2表达无明显变化(P>0.05).结论:Dlx2过表达上调AIP、OCN等成骨相关基因的表达.促进MC3T3-E1细胞的成骨分化.

Effects of Dlx2 overexpression on osteogenic differentiation of MC3T3-E1 cells

PURPOSE： To investigate the effect of Dlx2 overexpression on osteogenic differentiation of MC3T3-E1 cells in vitro.METHODS： Dlx2-expression retrovirus vector was constructed by subcloning and verified by sequencing.MC3T3-E1 cells were transfected with pMSCV-Dlx2 and selected with puromycin for stable clones.The expression of Dlx2 was determined by RT-PCR and Western blot.The level of osteogenetic biomarkers ALP,OCN,Runx2 and Msx2 was quantified by real-time PCR.ALP detection and Alizarin red staining were conducted to evaluate the effect of Dlx2 overexpression on osteogenic differentiation.The data were analyzed for ANOVA with SNK method using SAS 6.04 software package.RESULTS： pMSCV-Dlx2 was successfully constructed and verified by direct sequencing and Dlx2 overexpression in vitro.Enhanced ALP activity and Alizarin red staining were observed in MC3T3-E1-Dlx2 cells as compared with the control.During osteogenetic induction,Dlx2 overexpression up-regulated ALP and Msx2 in the early stage,while enhanced OCN expression in the late stage.Runx2 expression was different but not reached statistical significance.CONCLUSION： Dlx2 overexpression induces osteogenic differentiation of MC3T3-E1 cells via up-regulating bone formation-related genes.