RNA干扰survivin基因增强SKBr-3细胞对多柔吡星敏感性的体外研究  

RNA-interfered survivin gene increases sensitivity of SKBr-3 cells to doxorubicin:an in vitro study

作　　者：李庆霞[1] 王娟[1] 赵文清[1] 刘家云[2] 黄红艳[3] 张明[1] 杨安钢[4] 

机构地区：[1]河北省人民医院肿瘤一科,石家庄050051 [2]第四军医大学附属西京医院检验科,西安710032 [3]军事医学科学院附属307医院肿瘤三科,北京100071 [4]第四军医大学免疫学教研室,西安710032

出　　处：《第三军医大学学报》2010年第11期1158-1161,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30500433);河北省科技支撑计划(072761677)~~

摘　　要：目的探讨抑制survivin基因的表达对人乳腺癌SKBr-3细胞化疗敏感性的影响。方法构建针对survivin基因序列特异性shRNA的表达载体,脂质体转染SKBr-3,G418筛选阳性克隆。实验分为RNA干扰组(S1&P),脂质体对照组(H1&P)和空白对照组。光镜观察转染后细胞的形态变化;0.5μg/ml多柔吡星作用于转染后的细胞,电镜观察细胞形态学变化;TUNEL染色、AnnexinV荧光标记检测细胞凋亡,MTT法检测细胞增殖的抑制率。结果成功构建具有新霉素抗性的靶向survivin的序列特异性shRNA表达载体,基因测序完全正确;转染后干扰组细胞均发生细胞凋亡的形态学改变;低剂量多柔吡星诱导后,TUNEL染色结果 ,S1&P组凋亡细胞比例(23.6±4.8)%明显高于H1&P组(4.1±1.1)%和空白对照组(3.2±1.4)%(P<0.05),表明转染了RNA干涉载体的SKBr-3细胞对低剂量多柔吡星的敏感性显著增加;Annexin-V染色结果 :空白对照组凋亡比例为(17.43±3.12)%,H1&P组为(20.51±4.67)%,S1&P组为(68.76±8.49)%,后者与前两者比较均有统计学差异(P<0.05)。干扰组细胞凋亡率、细胞增殖的抑制率均明显增加,差异有统计学意义(P<0.05)。结论体外实验证实抑制survivin基因的表达可提高乳腺癌SKBr-3细胞对多柔吡星的敏感性。Objective To study the effect of RNA-interfered survivin gene on chemosensitivity of breast cancer cell line SKBr-3 to doxorubicin. Methods A shRNA expression vector,ΔpcDNA3-H1-S1,was constructed according to the specificity of survivin gene. SKBr-3 cells were transfected with liposome-encapsulated plasmids and positive clones were screened with G418. SKBr-3 cells were divided into RNA interference group,liposome control group and blank control group. Change of morphology in cells transfected with 0. 5 μg/ml doxorubicin was observed under an optical or electron microscope. Cell apoptosis was detected by TUNEL assay with An-nexin-Ⅴ fluorescence staining,and inhibiting rate of cell proliferation was detected by MTT assay. Results The survivin gene sequence specific shRNA expression vector resistant to neomycin was successfully constructed,which was confirmed by DNA sequencing. The morphologic change of apoptotic cells was observed in RNA inter-ference group. The rate of apoptotic cells was significantly higher in RNA interference group than in liposome control group and blank control group （ 23. 6 ±4. 8） % vs （ 4. 1 ±1. 1） % and （ 3. 2 ±1. 4） % after TUNEL staining （ P 0.05） ,indicating that the sensitivity of SKBr-3 cells to doxorubicin was increased in RNA-interfered vector. The rate of apoptotic cells was significantly lower in liposome control group and blank control group than in RNA interference group （ 17. 43 ± 3. 12） % and （ 20. 51 ± 4. 67） % vs （ 68. 76 ± 8. 49） % after Annexin-V staining （ P 0.05）. Conclusion The chemosensitivity of SKBr-3 cells to doxorubicin can be significantly increased by inhibiting the expression of survivin gene with RNA interference technology.

关 键 词：RNA干扰 SURVIVIN SKBR-3细胞 药物敏感性 

分 类 号：R73-362[医药卫生—肿瘤] R730.53[医药卫生—临床医学]