下调TSG101基因对人急性髓性白血病细胞的调节作用  

Effects of Downregulating TSG101 on the Phenotype of Human Leukemia Cells

作　　者：刘丽华[1] 张宏卫[2] 弭忠祥[3] 

机构地区：[1]绍兴文理学院医学院分子诊断实验室,绍兴312000 [2]绍兴文理学院医学院附属医院神经科,绍兴312000 [3]绍兴文理学院生命科学院,绍兴312000

出　　处：《细胞生物学杂志》2006年第4期622-626,共5页Chinese Journal of Cell Biology

基　　金：浙江省自然科学基金资助项目(No.302301)~~

摘　　要：构建肿瘤易感基因101(TSG101)基因的小干扰RNA载体并将其转导入HL-60细胞,获得稳定转染的阳性克隆后,应用RT-PCR和Western印迹进行鉴定;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;DNA梯带(ladder)法和流式细胞仪检测细胞对顺铂诱导的凋亡的变化;Western印迹检测耐药相关蛋白P-gp和MRP的表达变化。结果表明,成功构建了TSG101的小干扰RNA载体;经蛋白质水平检测证实成功建立了稳定的TSG101低表达的白血病细胞模型;转染TSG101小干扰RNA后的细胞生长速度显著减慢,出现G1期阻滞;对顺铂的敏感性明显增强,形成DNA条带,且低表达P-gp。因此,下调TSG101基因能抑制HL-60细胞生长,增加细胞对化疗药物的敏感性,并提示该基因具有进行白血病基因治疗的可行性。The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination, then transfected into HL-60 cells. Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blot analysis. The growth curve was made using MTT assay. Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferous indexes were calculated. The apoptosis after CDDP treatment was detected by DNA ladder and annexin V/propidium iodide binding analyses. The expressions of P-gp and MRP were analyzed by Westem blot. mU6pro-TSG101 siRNA was successfully constructed and transfected into HL-60 cells. Down-regulation of TSG101 could significantly sup- press the proliferation of HL-60 cells with a G1 cell cycle arrest, and enhance the sensitivity of HL-60 cells towards CDDP-induced apoptosis. The expressions of P-gp in transfected cells was decreased as compared with that of the control, while MRP not. Therefore, down-regulation of TSG 101 could suppress the proliferation of HL-60 cells, and enhance the sensitivity of HL-60 cells to conventional chemotherapeutic agents to a degree, suggesting TSG101 could be used for gene therapy in future.

关 键 词：白血病 TSG101 RNA干扰 

分 类 号：R733.71[医药卫生—肿瘤]