文献类型：期刊文献

中文题名：TSG101小干扰RNA对SH-SY5Y细胞生长和药物敏感性的作用

英文题名：Effects of TSG101 siRNA on the growth and drug sensitivity of SH-SY5Y cells

作者：刘丽华[1];张宏卫[2];王承忠[3]

机构：[1]绍兴文理学院医学院分子诊断实验;[2]绍兴文理学院医学院附属医院神经科;[3]中国科学院神经科学研究所

年份：2006

卷号：22

期号：9

起止页码：1734

中文期刊名：中国病理生理杂志

外文期刊名：Chinese Journal of Pathophysiology

收录：CSTPCD、、北大核心2004、CSCD2011_2012、北大核心、CSCD

语种：中文

中文关键词：神经母细胞瘤;基因,TSG101;RNA干扰;SH-SY5Y细胞

外文关键词：Neuroblastoma; Genes, TSG101 ; RNA interference; SH-SY5Y cells

中文摘要：目的:探讨TSG101基因对人神经母细胞瘤细胞生长和药物敏感性的调节作用。方法:构建TSG101基因的小干扰RNA载体并将其转导入SH-SY5Y细胞,G418筛选后获得稳定转染的阳性克隆后,应用RT-PCR和Western blotting进行鉴定;MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化;DNAlad-der法和流式细胞仪检测细胞转染前后对顺铂诱导的凋亡的变化;Western blotting检测细胞转染前后凋亡相关蛋白Bcl-2、Bax,耐药相关蛋白P-gp、MRP的表达变化。结果:成功构建了TSG101的小干扰RNA载体并将其转染SH-SY5Y细胞;筛选到稳定的TSG101低表达的神经母细胞瘤细胞模型;MTT和流式细胞仪检测结果显示,转染TSG101小干扰RNA后的细胞的生长速度显著减慢于对照组(P<0·05),且G1期的细胞比率显著高于对照组(P<0·05);DNAladder法和流式细胞仪检测结果显示,用10mg/LCDDP处理36h后,转染TSG101小干扰RNA后的细胞的凋亡数显著多于对照组(P<0·05),形成DNA条带;Western blotting显示,转染TSG101小干扰RNA后的细胞中Bcl-2和P-gp的表达明显低于对照组。结论:下调TSG101基因能抑制神经母细胞瘤细胞生长,增加细胞对化疗药物的敏感性,提示TSG101在基因治疗中具有较好的临床应用前景。

外文摘要：AIM： To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human neuroblastoma cell line SH -SY5Y. METHODS： The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination, then transfected into SH -SY5Y cells. Stable transfectants were obtained by G418 screening and further identified by RT - PCR and Western blotting analysis. The growth curve was made using MTT assay. Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferative indexes were calculated. The apoptosis after CDDP treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses. The expression of Bcl - 2, Bax, P - gp and MRP were analyzed by Western blotting. RESULTS ： mU6pro - TSG101 siRNA was successfully constructed and transfected into SH -SY5Y cells. As detected by MTT and flow cytometry, down- regulation of TSG101 significantly suppressed the proliferation of SH -SY5Y cells with a G1 cell cycle arrest, compared with that in control （ P 〈 0.05 ）. As detected by DNA ladder and Annexin V/propidium iodide binding analyses, down - regulation of TSG101 significantly enhanced the sensitivity of SH - SY5Y cells to CDDP - induced apoptosis, compared with that in control （ P 〈 0. 05 ）. The expression of P - gp and Bcl - 2 in transfected cells were decreased as compared with that in the control, while MRP and Bax were not. CONCLUSIONS： Down -regulation of TSG101 suppresses the proliferation of SH - SY5Y cells, and enhances the sensitivity of SH - SY5Y cells to conventional chemotherapeutic agents to a degree, suggesting TSGIOI may be useful for gene therapy in the future.