Bcl-2基因表达抑制联合射线对NCI-H460细胞增殖状态及细胞周期影响的研究

目的 利用RNAi技术抑制Bcl-2基因的表达,联合放疗后观察其对非小细胞肺癌NCI-H460株细胞周期及细胞增殖状态的影响。方法 利用以pCYU6/GFP/Neo为载体的Bcl-2/shRNA重组质粒转染非小细胞肺癌NCI-H460细胞株,采用Western blot方法检测Bcl-2基因蛋白表达水平的变化,继而给予直线加速器6 Gy X线照射,采用MTT法和流式细胞术检测RNA干扰及照射后对NCI-H460细胞增殖和细胞周期的影响。结果 成功转染并经筛选获得阳性转染成功的细胞(HT),荧光显微镜下可见发绿色荧光的NCI-H460细胞,证明转染成功;Western blot检测显示Bcl-2蛋白表达水平显著降低;X线照射6 Gy,24 h及48 h后转染有义序列组细胞增殖情况较无义序列组及未转染组明显降低;流式细胞术显示转染有义序列细胞组经照射后24小时G₂/M期分布比例增高,48小时后G₂/M期分布比例显著减低。结论 RNAi技术可以有效的抑制非小细胞肺癌NCI-H460细胞Bcl-2基因表达,联合放疗后NCI-H460细胞的增殖率及细胞周期发生改变,起到一定的放射增敏作用。

To Explore the Activity of Generation and the Cell Cycle of NCI-H460 by RNAi Inhibiting Bcl-2 Gene Expression and Combining Treatment with Radiation

Objective To explore the influence of Bcl - 2 gene expression by RNAi on cell cycle and activity of cell and the radiosensitivity of Non - Small cell lung cancer cell line NCI - 1-1460. Methods Making use of pCYU6/GFP/Neo on vectors containing shRNA targeting Bcl - 2 gene were transfeeted into NCI - H460. Western blot were used to compare the efficiency of gene silencing. The transfected cell were exposed to 6 Gy X - rays generated by a linear accelerator. MTT and Flow cytometry were used to explore the activity of generation and the cell cycle of NCI - H460. Results Bcl - 2/shRNA was successfully transfected NCI - I-I460 cell lines. Western blot analysis that expression of Bcl -2 was suppressed by Bcl -2/shRNA;The activity of generation of cells transfected with Bcl- 2/shRNA was lower than others at 6 Gy irradiation, and higher percentage of GJM phase was observed on cells transfected with Bcl -2/shRNA than others by cytometry. Conclusion RNAi could inhibit Bcl- 2 gene expression and enhance the activity of generation and the cell cycle of NCl - Hd60 cells.