沉默PeroxiredoxinⅠ基因增强乳腺癌细胞放射-第三军医大学学报.DOC

沉默PeroxiredoxinⅠ基因增强乳腺癌细胞放射敏感性及其作用机制的实验研究 郭启帅1，黄 曦2，李少林1 (400016 重庆,重庆医科大学放射医学教研室1；401520重庆，重庆市合川区人民医院肿瘤血液科2) [摘要] 目的 采用RNA干扰技术沉默过氧化物酶Ⅰ（PeroxiredoxinⅠ，PrxⅠ）基因，观察乳腺癌MCF-7细胞对X线敏感性变化,探讨其作用机制。方法 将PrxⅠshRNA真核表达载体pGPU-PrxⅠ和阴性对照质粒pGPU6-HK转染入乳腺癌MCF-7细胞中，经G418稳定筛选后RT-PCR和Western blot检测PrxⅠ表达变化。6 MV X线照射6 Gy后48 h,流式细胞术检测细胞的活性氧水平、细胞周期和细胞凋亡；MTT法测定细胞增殖能力；克隆形成实验观察克隆形成率，计算Do、N、Dq、SF2及放射增敏比(sensitive enhancement ratio，SER)等放射生物学参数，Western blot法检测Rad51及γ-H2AX蛋白变化。结果 获得2个稳定转染细胞株：pGPU6-HK和pGPU6-PrxⅠ。RT-PCR和Western bolt检测显示pGPU6-PrxⅠ组中PrxⅠmRNA和蛋白表达均明显抑制。6 Gy的X线照射48 h后，pGPU-PrxⅠ及联合照射（Ionizing radiation，IR）组细胞中的活性氧水平、G1期细胞和细胞凋亡明显增加，而细胞增殖能力和S期细胞明显降低，Western blot分析表明Rad51蛋白表达降低，而γ-H2AX蛋白表达升高(P＜0.05)，以pGPU-PrxⅠ+IR组变化最显著，其Rad51蛋白表达下降了79.3%，而γ-H2AX蛋白表达升高了3.7倍。pGPU-PrxⅠ组细胞的Do、N、Dq和SF2值分别为1.354、3.106、1.547和0.504，均低于pGPU-HK组,SF2时的SER为1.353。结论 下调PrxⅠ基因表达可提高乳腺癌MCF-7细胞的放射敏感性，其作用机制与细胞内活性氧清除能力减弱、DNA双链断裂增加及DNA损伤后修复能力降低有关。PrxⅠ可能是一个理想的肿瘤放射增敏分子靶点。 [关键词] PeroxiredoxinⅠ；放射敏感性；活性氧 [中图法分类号] R737.9, R730.55 [文献标志码] A Effect of silencing PeroxiredoxinⅠ gene by RNA interference on the radiosensitivity of breast carcinoma cell line MCF-7 and explore its mechanism Guo Qishuai1, Huang Xi 2, Li Shaolin1 (1.Department of Radiation Medicine，Chongqing Medical University, Chongqing, 400016; 2. Department of Oncology and Hematology，Hechuan District Peoples’s Hospital, Chongqing，400016, China) [Abstract] 0bjective: To investigate the influence of silencing PrxⅠgene by RNA interference on the radiosensitivity of breast carcinoma cell line MCF-7 and its mechanism. Methods:The recombinant plasmids pGPU6-HK and pGPU-PrxⅠhad successfully constructed were transfected into MCF-7 cell by Lipofectamine respectively. The mRNA and protein expressions of PrxⅠwere detected by RT-PCR and Western blot. The Reactive oxygen species(ROS), cell cycle and cell apoptosis were estimated by Flow Cytometry, .the cell proliferation was determined by MTT assay and the protein expressions of Rad51 and γ-H2AX were evaluated by Western Blotting