目的： 研究siRNA沉默毛细血管扩张—共济失调突变（atxia-telangiectasia mutated，ATM）基因的表达增强胞嘧啶鸟嘌呤二核苷酸寡脱氧核苷酸(cytosine-phophate-guanine oligodeoxynucleotide，CpG ODN) 7909对人非小细胞肺癌A549细胞的放射增敏作用。 方法: 将ATM-siRNA转染至A549细胞中，Western blotting检测A549细胞中ATM蛋白的表达。A549细胞随机分为6组：对照组、CpG组、X射线（IR）组、CpG+IR组、ATM-siRNA+CpG+IR组和NC-siRNA+CpG+IR组，克隆形成分析法检测各组细胞克隆形成率，Graphpad prism 5.0软件进行单击多靶模型和L-Q 线性模型拟合辐射后A549细胞的生存曲线，以D0、Dq、N、α/β及SF2等参数分析A549细胞辐射损伤修复能力，流式细胞术检测A549细胞的凋亡。 结果: ATM-siRNA转染可明显抑制A549细胞中ATM蛋白的表达（P<0.01）。X射线可剂量依赖性抑制A549细胞的克隆形成能力（P<0.05）；且CpG+IR组A549细胞的克隆形成能力进一步降低（P<0.01）；ATM-siRNA转染后，CpG处理的A549细胞克隆形成能力再度降低\[10 Gy时，（0.05±0.00）% vs （0.71±000）%，P<0.01\]。辐射损伤剂量生存曲线结果显示，ATM-siRNA转染后，ATM-siRNA+CpG+IR组较CpG+IR组A549细胞的α/β值明显增大（1.48 vs 0.97，P<0.05），对放射损伤修复能力明显减弱。CpG+IR组较IR组细胞凋亡率显著升高\[（9.18±0.16）% vs （6.56±0.33）%，P<0.01\]； ATM-siRNA+CpG+IR组A549细胞凋亡率进一步升高\[（10.45±0.40）% vs （9.18±0.16）%，P<0.05\]。 结论： siRNA 沉默ATM 的表达可增强CpG ODN 7909对A549细胞的放射增敏作用，ATM可作为肺癌治疗的潜在靶点。

Objective:To explore the potentiation of silencing atxia-telangiectasia mutated (ATM) gene expression in the radiosensitization effect of cytosine-phophate-guanine oligodeoxynucleotide (CpG ODN) 7909 on non-small cell lung cancer A549 cell line. Methods: ATM-siRNA was transfected into A549 cells, and the expression of ATM protein in A549 cells was examined by Western blotting. A549 cells were randomly classified into six groups: control group, CpG group, X-ray (IR) group, CpG+IR group, ATM-siRNA+CpG+IR group and NC-siRNA+CpG+IR group. Cell colony rates were evaluated by colony formation assay. One-hit multi-target model and linear quadratic model, which generated the radiation dose survival curve of the A549 cells, were fitted with Graphpad prism 5.0 software, and the parameters, including D0, Dq, N, α/β and SF2 were applied to analyze radiation damage repair capacity of A549 cells. The apoptosis of A549 cells was analyzed by flow cytometry. Results: ATM-siRNA transfection remarkably inhibited the expression of ATM protein in A549 cells (P<001). X-ray radiation inhibited the colony formation capacity of A549 cells in a dose-dependent manner (P<0.05). Moreover, a further decrease of the colony formation capacity of A549 cells was found in CpG+IR radiation combined treatment group (P<0.01). With the transfection of ATM-siRNA, the colony formation capacity of CpG-treated A549 cells was further decreased (10 Gy, \[0.05±0.00\]% vs \[0.71±0.00\]%, P<0.01). Radiation damage dose survival curves demonstrated that the value of α/β was significantly increased (1.48 vs 0.97，P<005)and the radiation damage repair capability was significantly decreased in ATM-siRNA+CpG+IR group compared with CpG+IR group. The apoptotic rate was significantly increased in CpG+IR group compared with IR radiation group (\[9.18±016\]% vs \[6.56±0.33\]%, P<0.01). The apoptotic rate of A549 cells in ATM-siRNA+CpG+IR group was further increased (\[9.18±0.16\]% vs \[10.45±0.40\]%, P<0.05). Conclusion:Silencing expression of ATM by siRNA can enhance the radiosensitization effect of CpG ODN 7909 on A549 cells. ATM may serve as a potential target for gene therapy of lung cancer.

上海金山区科技创新基金资助项目( No. 2010-316 )