肿瘤坏死因子受体相关因子6基因沉默对成牙本质细胞增殖能力的影响

Effect of tumor necrosis factor receptor associated factor 6 gene silencing by small interfering RNA on proliferation of odontoblast cells

目的 研究小干扰RNA （small interfering RNA,siRNA）抑制肿瘤坏死因子受体相关因子6 （tumor necrosis factor receptor associated factor 6,TRAF6）的表达后,对小鼠成牙本质细胞样细胞MDPC-23增殖能力的影响,以期构建TRAF6基因沉默的成牙本质细胞模型,并阐明TRAF6对成牙本质细胞增殖的调控作用.方法 将针对TRAF6基因的siRNA真核表达载体pSUPER-TRAF6siRNA转染MDPC-23（转染组）,空载体对照组采用pSUPER空载体质粒,空白对照组MDPC-23细胞不转染任何质粒,采用氨基糖苷类抗生素G418抗性筛选,挑选克隆株;反转录PCR （reverse transcription-PCR,RT-PCR）和蛋白质印迹法检测TRAF6的mRNA和蛋白表达;通过绘制细胞生长曲线、甲基噻唑基四唑（methyl thiazolyl tetrazolium,MTr）、流式细胞仪（flow cytometry,FCM）检测转染前后细胞增殖能力的变化.结果 MDPC-23细胞转染pSUPER-TRAF6siRNA后,可以稳定表达克隆株,其TRAF6mRNA和蛋白表达水平与两个对照组相比均显著下降[mRNA：转染组为0.163±0.008,空载体对照组为0.778±0.017,空白对照组为0.782±0.004,P＜0.000 1;蛋白质印迹法：转染组为0.215±0.006,空载体对照组为0.964±0.007,空白对照组为0.973±0.013,P＜0.001];稳定转染TRAF6siRNA的单克隆株3、5d时,转染组A值为0.46±0.03和1.35±0.06,均显著高于相同时间点的空载体对照组和空白对照组（P＜0.01）;转染组的细胞增殖指数（proliferation index,PrI）[（24.1±2.2）％]均显著高于空载体对照组[（11.2±1.0）％]和空白对照组[（10.5±0.7）％]（P＜0.01）.结论 稳定转染pSUPER-TRAF6siRNA后可以显著降低MDPC-23细胞中TRAF6的表达,表明构建TRAF6基因沉默的成牙本质细胞模型成功;TRAF6基因沉默可以使MDPC-23细胞的增殖能力增强,将影响成牙本质细胞形成和修复牙本质的能力,表明TRAF6是调控牙本质发育及损伤修复过程的重要信号分子.

Objective To investigate the effect of small interfering RNA（siRNA）-mediated inhibition of tumor necrosis factor receptor associated factor 6（TRAF6） gene on murine odontoblast-like cell line,MDPC-23 cell and the effect of TRAF6 on MDPC-23 cell proliferation.Methods The vectors expressing siRNA against TRAF6 were constructed and introduced into MDPC-23 cell with lipofectin,and the cell line with stable expression of siRNA of TRAF6 was obtained by G418 screening and colony culture.Reverse transcription-PCR（RT-PCR） and Western blotting were performed to detect the expression of TRAF6.The proliferation of transfected MDPC-23 cell was investigated through methabenzthiazuron（MTF）and flow cytometry（FCM） assay.Results The positive single colony was screened out,and was found to express siRNA against TRAF6 effectively because both TRAF6 mRNA and protein relative expression were significantly decreased in the experimental group（pSUPER-TRAF6siRNA：mRNA 0.163 ± 0.008,protein 0.215±0.006） compared with controls（pSUPER：mRNA 0.778±0.017,protein 0.964±0.007 （P 〈 0.001）.The A value of treated pSUPER-TRAF6siRNA cells （3 d：0.46±0.03,5 d：1.35±0.06） was increased compared with controls（P〈0.01）.The result of proliferation index（PrI） was also increased compared with controls [pSUPER-TRAF6siRNA：（24.1 ± 2.2）% ; pSUPER（11.2 ± 1.0）% ; control（10.5 ± 0.7）%,P〈0.01].Conclusions The transcription and expression of TRAF6 gene were inhibited.The proliferation ability was increased in MDPC-23 cells by the constructed pSUPER-TRAF6siRNA vector.It may further influence the formation and repair of dentin,and may be involved in the regulation of normal tooth eruption and process of dentin repair after injury.