【摘要】 目的用人工合成的耐药相关基因细胞周期蛋白D1(cyclinD1)的小分子干扰RNA(siRNA)处理人舌鳞癌顺铂耐药细胞系(Tca8113-CDDP),探讨对靶基因转录调控的作用效果以及剂量和时间效应关系,为耐药性逆转提供实验依据。方法CY3荧光素标记阳性对照基因甘油醛-3-磷酸脱氢酶(GAPDH)siRNA,分别于转染后4、24、48、72h在激光共聚焦显微镜下观察瞬时转染率,优化转染条件。用SYBR(绿色荧光染料法、实时定量PCR技术分析目的基因mRNA的表达情况,同时采用蛋白印迹(Westernblot)检测目的基因蛋白水平的表达。四甲基偶氮唑盐法检测cyclinD1siRNA处理后细胞对顺铂敏感性的影响。结果优化转染条件后,Tca8113-CDDP细胞中CY3的GAPDHsiRNA转染率达90%以上。转染cyclinD1siRNA后24、48hmRNA表达的抑制率为81.6%、80.7%,72h最为明显,为94.3%。在低于100nmol/L浓度下,RNA干扰抑制与剂量大小有关;当剂量进一步加大时,抑制效应则维持在“平台期”。转染24、48和72h后,cyclinD1蛋白的表达也明显下降。siRNA干扰组和对照组细胞在顺铂作用后细胞生长抑制率分别为58.4%、34.8%。结论化学合成的cyclinD1siRNA在体外实验中,对Tca8113-CDDP细胞中目的基因的沉默提高了细胞对顺铂敏感性,并呈现剂量和时间依赖效应。更多还原

【Abstract】 Objective To interfere in the Tca8113-CDDP cell line with siRNA of cyclin D1 and to investigate time and dose dependent gene silencing effect of siRNA of cyclin D1.Methods siRNA of cyclin D1 was transfected into Tca8113-CDDP cells Fluorescent CY3 dye labeled siRNA GAPDH was used as the control. The transient transfecting efficiency was examined at 4, 24, 48 and 72 h. The relative quantity of the target RNA of cyclin D1 was analyzed with SYBR Green fluorescent dye kit by the Real-time PCR assay. The protein level of cyclin D1 was examined with Western blot. The changes of cisplatin sensitivity after treatment with siRNA cyclin D1 were examined with methyl thiazolyl teerazolium(MTT) assay.Results The optimized transfecting efficiency with CY3 labeled siRNA GAPDH in Tca8113-CDDP cells was over 90%. The silencing rate of cyclin D1 siRNA was 81.6% at 24 h, 80.7% at 48 h and 94.3% at 72h. Dose-dependent manner of gene silencing effect was observed when the siRNA concentration was lower than 100 nmol/L , however, gene silencing effect reached its platform when the concentration was higher than 100 nmol/L . The protein levels of cyclin D1 at 24, 48 and 72 h after transfection decreased significantly,and so did the growth of cells. Inhibition rates of cell growth induced by cisplatin after administration with or without cyclin D1 siRNA were 58.4% and 34.8%, respectivly.Conclusions Chemical synthesized cyclin D1 siRNA effectively silenced the expression of cyclin D1 gene in Tca8113-CDDP cells in vitro, with a time-and dose-dependent manner and target gene silence in cells increased its sensitivity to cisplatin.更多还原