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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 investigate the effect of inhibiting the expression of survivin gene on the chemosensitivity of human breast cancer SKBr-3 cells. Methods The expression vector targeting shRNA targeting survivin gene was constructed and transfected into SKBr-3 cells by Lipofectamine 2000. The positive clones were screened by G418. The experiment was divided into RNA interference group (S1 & P), liposome control group (H1 & P) and blank control group. Morphological changes of the cells were observed with light microscope; 0.5μg / ml doxorubicin was applied to the transfected cells, the morphological changes of the cells were observed by electron microscope; Cell apoptosis was detected by TUNEL staining and Annexin V fluorescent labeling; Cell proliferation was detected by MTT assay; Inhibition rate. Results The sequence-specific shRNA expression vector targeting survivin with neomycin resistance was successfully constructed and the gene sequencing was completely correct. The morphological changes of apoptotic cells were observed in the transfected cells after transfection. After induction with low dose of doxorubicin (23.6 ± 4.8)% in S1 & P group was significantly higher than that in H1 & P group (4.1 ± 1.1%) and control group (3.2 ± 1.4)% (P <0.05) The sensitivity of SKBr-3 cells to low doses of doxorubicin significantly increased. The results of Annexin-V staining showed that the percentage of apoptotic cells in the control group was (17.43 ± 3.12)%, that in the control group was (20.51 ± 4.67)%, and that in the S1 & P Group (68.76 ± 8.49)%, the latter with the former two were statistically significant difference (P <0.05). The rates of apoptosis and cell proliferation were significantly increased in the interference group (P <0.05). Conclusion In vitro experiments show that inhibiting survivin gene expression can improve the sensitivity of breast cancer SKBr-3 cells to doxorubicin.