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目的利用RNAi技术抑制Bcl-2基因的表达,观察其对肺腺癌A549细胞凋亡和放射敏感性变化的影响。方法构建Bcl-2基因的干扰质粒pBcl-2-siRNA,稳定转染人肺腺癌A549细胞,同时设立转染无意义链组、空载体组和未转染组。采用Real time RT-PCR和Western blot方法,观察Bcl-2基因在mRNA和蛋白水平表达的变化。采用流式细胞术和克隆形成方法,检测RNA干扰对A549细胞放射敏感性的影响。结果成功构建bcl-2 siRNA质粒,转染A549细胞,获得稳定转染细胞株A549/Bcl2-Ins;Bcl-2mRNA、Bcl-2蛋白表达明显降低;A549/Bcl2-Ins凋亡率[(10.11±0.92)%]明显高于对照组,6Gy X线照射后凋亡率[(28.81±1.10)%]明显高于对照组及未照射组;A549/Bcl2-Ins的D0和Dq值分别为1.399和1.298,明显低于转染无意义序列细胞株A549/Bcl2-Neg(1.695和2.480)、转染空载体细胞株A549/SD(1.706和2.227)和空白对照细胞A549(1.816和2.777)。结论RNAi技术可以有效抑制肺腺癌A549细胞Bcl-2基因的表达,增强A549细胞对X线的放射敏感性。
Objective To inhibit the expression of Bcl-2 gene by RNA interference (RNAi) and observe its effect on the apoptosis and radiosensitivity of lung adenocarcinoma A549 cells. Methods Plasmid pBcl-2-siRNA targeting Bcl-2 gene was constructed and stably transfected into human lung adenocarcinoma A549 cells. At the same time, the vector was transfected into the nonsense vector, empty vector and untransfected A549 cells. Real time RT-PCR and Western blot were used to observe the changes of Bcl-2 mRNA and protein expression. The effects of RNA interference on radiosensitivity of A549 cells were detected by flow cytometry and clonogenic assay. Results Bcl-2 siRNA plasmid was successfully constructed and A549 / Bcl2-Ins was transfected into A549 cells. The expression of Bcl-2 and Bcl-2 mRNA was significantly decreased. The apoptosis rate of A549 / Bcl2- 0.92)%] was significantly higher than that of the control group. The apoptotic rate after irradiation with 6Gy X-rays [(28.81 ± 1.10)%] was significantly higher than that of the control group and the non-irradiated group. The D0 and Dq values of A549 / Bcl2- 1.298, which was significantly lower than that of transfected A549 / Bcl2-Neg (1.695 and 2.480). The transfected A549 / SD (1.706 and 2.227) cells and blank control A549 cells (1.816 and 2.777) were transfected. Conclusion RNAi can effectively inhibit the expression of Bcl-2 gene in lung adenocarcinoma A549 cells and enhance the radiosensitivity to A549 cells.