中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2014年
7期
428-433
,共6页
李霞%肖明振%余擎%赵彬%李卫星
李霞%肖明振%餘擎%趙彬%李衛星
리하%초명진%여경%조빈%리위성
RNA干扰%转染%细胞增殖%肿瘤坏死因子受体相关因子6
RNA榦擾%轉染%細胞增殖%腫瘤壞死因子受體相關因子6
RNA간우%전염%세포증식%종류배사인자수체상관인자6
RNA interfering%Transfection%Cell proliferation%Tumor necrosis factor receptor associated factor 6
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.
目的 研究小榦擾RNA (small interfering RNA,siRNA)抑製腫瘤壞死因子受體相關因子6 (tumor necrosis factor receptor associated factor 6,TRAF6)的錶達後,對小鼠成牙本質細胞樣細胞MDPC-23增殖能力的影響,以期構建TRAF6基因沉默的成牙本質細胞模型,併闡明TRAF6對成牙本質細胞增殖的調控作用.方法 將針對TRAF6基因的siRNA真覈錶達載體pSUPER-TRAF6siRNA轉染MDPC-23(轉染組),空載體對照組採用pSUPER空載體質粒,空白對照組MDPC-23細胞不轉染任何質粒,採用氨基糖苷類抗生素G418抗性篩選,挑選剋隆株;反轉錄PCR (reverse transcription-PCR,RT-PCR)和蛋白質印跡法檢測TRAF6的mRNA和蛋白錶達;通過繪製細胞生長麯線、甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTr)、流式細胞儀(flow cytometry,FCM)檢測轉染前後細胞增殖能力的變化.結果 MDPC-23細胞轉染pSUPER-TRAF6siRNA後,可以穩定錶達剋隆株,其TRAF6mRNA和蛋白錶達水平與兩箇對照組相比均顯著下降[mRNA:轉染組為0.163±0.008,空載體對照組為0.778±0.017,空白對照組為0.782±0.004,P＜0.000 1;蛋白質印跡法:轉染組為0.215±0.006,空載體對照組為0.964±0.007,空白對照組為0.973±0.013,P＜0.001];穩定轉染TRAF6siRNA的單剋隆株3、5d時,轉染組A值為0.46±0.03和1.35±0.06,均顯著高于相同時間點的空載體對照組和空白對照組(P＜0.01);轉染組的細胞增殖指數(proliferation index,PrI)[(24.1±2.2)％]均顯著高于空載體對照組[(11.2±1.0)％]和空白對照組[(10.5±0.7)％](P＜0.01).結論 穩定轉染pSUPER-TRAF6siRNA後可以顯著降低MDPC-23細胞中TRAF6的錶達,錶明構建TRAF6基因沉默的成牙本質細胞模型成功;TRAF6基因沉默可以使MDPC-23細胞的增殖能力增彊,將影響成牙本質細胞形成和脩複牙本質的能力,錶明TRAF6是調控牙本質髮育及損傷脩複過程的重要信號分子.
목적 연구소간우RNA (small interfering RNA,siRNA)억제종류배사인자수체상관인자6 (tumor necrosis factor receptor associated factor 6,TRAF6)적표체후,대소서성아본질세포양세포MDPC-23증식능력적영향,이기구건TRAF6기인침묵적성아본질세포모형,병천명TRAF6대성아본질세포증식적조공작용.방법 장침대TRAF6기인적siRNA진핵표체재체pSUPER-TRAF6siRNA전염MDPC-23(전염조),공재체대조조채용pSUPER공재체질립,공백대조조MDPC-23세포불전염임하질립,채용안기당감류항생소G418항성사선,도선극륭주;반전록PCR (reverse transcription-PCR,RT-PCR)화단백질인적법검측TRAF6적mRNA화단백표체;통과회제세포생장곡선、갑기새서기사서(methyl thiazolyl tetrazolium,MTr)、류식세포의(flow cytometry,FCM)검측전염전후세포증식능력적변화.결과 MDPC-23세포전염pSUPER-TRAF6siRNA후,가이은정표체극륭주,기TRAF6mRNA화단백표체수평여량개대조조상비균현저하강[mRNA:전염조위0.163±0.008,공재체대조조위0.778±0.017,공백대조조위0.782±0.004,P＜0.000 1;단백질인적법:전염조위0.215±0.006,공재체대조조위0.964±0.007,공백대조조위0.973±0.013,P＜0.001];은정전염TRAF6siRNA적단극륭주3、5d시,전염조A치위0.46±0.03화1.35±0.06,균현저고우상동시간점적공재체대조조화공백대조조(P＜0.01);전염조적세포증식지수(proliferation index,PrI)[(24.1±2.2)％]균현저고우공재체대조조[(11.2±1.0)％]화공백대조조[(10.5±0.7)％](P＜0.01).결론 은정전염pSUPER-TRAF6siRNA후가이현저강저MDPC-23세포중TRAF6적표체,표명구건TRAF6기인침묵적성아본질세포모형성공;TRAF6기인침묵가이사MDPC-23세포적증식능력증강,장영향성아본질세포형성화수복아본질적능력,표명TRAF6시조공아본질발육급손상수복과정적중요신호분자.
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.
