摘要
目的探讨用小RNA干扰抑制Ku80基因表达以提高A549肺癌细胞的放射敏感性。方法设计并化学合成Ku80siRNA,转染体外培养的A549肺癌细胞。利用RT-PCR和Western blot分别从mRNA和蛋白质水平对转染后的细胞Ku80基因表达情况进行测定,同时上述转染的细胞分别照射2、4、6、8及10Gy,利用克隆形成实验测定放射敏感性的变化。结果 RT-PCR检测显示在A549肺癌细胞转染Ku80siRNA后24、48和72h三个时间点Ku80mRNA含量减少,Western blot分析显示在转染48和72h两个时间点Ku80蛋白含量减少,与对照组比较有统计学差异(P<0.05)。克隆形成实验提示A549肺癌细胞转染Ku80siRNA后放射敏感性增强。结论 Ku80siRNA转染A549肺癌细胞后能够有效抑制Ku80基因表达,提高其放射敏感性。
Objective To investigate the radiation-sensitizing effects of Ku80 silencing by siRNA interference for A549 lung cancer cells.Methods The sequences of Ku80 siRNA and negative siRNA were chemically synthesized and transfected into A549 lung cancer cells by lipofectamine.RT-PCR and Western bolt analysis were used to determine Ku80 gene expression.The transfected cells in culture dishes were irradiated with X ray at doses of 2 Gy,4 Gy,6 Gy,8 Gy,10 Gy,respectively.Once all treatments were completed,the cells were processed with the colony formation assay.Results RT-PCR detection showed that Ku80 mRNA levels in A549 lung cancer cells were reduced after transfected with Ku80 siRNA at 24 h,48 h and 72 h time points.Western blot analysis showed that Ku80 protein content decreased at 48 h and 72 h time points compared with the control group （P0.05）.Cloning formation assay indicated that radiosensitivity of A549 lung cancer cells was enhanced after transfected with Ku80 siRNA.Conclusion Ku80 siRNA can effectively inhibit Ku80 gene expression of A549 lung cancer cells,and therefore enhance its radiosensitivity.
出处
《中国呼吸与危重监护杂志》
CAS
2010年第3期291-294,共4页
Chinese Journal of Respiratory and Critical Care Medicine
基金
卫生部科研基金资助项目(编号:wkj2005-2-051)
杭州市科技局资助项目(编号:20080333B10)
