环巴胺选择性增加肺癌细胞放射增敏的研究

摘要

目的 研究环巴胺对肺癌细胞和正常肺上皮细胞的放射敏感性的影响.方法 采用MTT测定细胞的生长和存活率;采用克隆形成实验观察环巴胺对肺癌细胞放射敏感性的影响;蛋白免疫印迹法检测Shh蛋白的表达;siRNA转染NIH-H226细胞观察降低Shh的表达对放射敏感性的影响.结果 MTT结果显示2.5 μmol/L环巴胺24 h处理对本实验研究的所有肺癌细胞系和正常肺上皮细胞系的生长和增殖未见明显的影响.采用2.5 μmol/L剂量的环巴胺预处理明显增加了NIH-H520、NIH-H1437、NIH-H1975、A549和NIH-H226 5种人肺癌细胞的放射敏感性,而并不影响IMR90、CCD-16Lu和BEAS-2B 3种正常肺上皮细胞的放射敏感性.在5种肺癌细胞系中可以检测到Shh蛋白不同程度的表达,而3种正常肺上皮细胞无Shh蛋白表达.Shh-siRNA转染的NIH-H226细胞的放射敏感性明显提高.结论 环巴胺可以降低肺癌细胞中的Shh蛋白表达水平.采用Shh-siRNA明显降低肺癌细胞中Shh蛋白表达,增加了细胞的放射敏感性,但抑制了环巴胺的放射增敏疗效.本实验结果证实环巴胺可以选择性提高肺癌细胞的放射治疗敏感性,调节Shh表达可能是其重要的机制之一.%Objective To study the impact of cyclopamine on radiosensitivity in lung cancer cell lines and normal lung epithelial cells. Methods MTT assay was used to determine cell growth and survival; clonogenic assay was employed to detect the impact of cyclopamine on the radiosensitivity of lung cancer cells; Western blot assay was employed to measure protein expression; Shh-siRNA was transfected into NIH-H226 cells to observe the influence of the Shh expression decrease on the radiosensitivity. Results 2.5 u,mol/L of cyclopamine showed no significant influence on the growth and proliferation of both lung cancer cells and normal lung epithelial cells. However,the dose of 2.5 |xmol/L significantly increased radiosensitivity of five human lung cancer cell lines,including NIH-H520,NIH-H1437,NIH-H1975,A549 and NIH-H226,but failed to affect radiosenstivity of three normal lung epithelial cell lines,including IMR90,CCD-16Lu and BEAS-2B. After the transfection of Shh-siRNA, the radiosensitivity of NIH-H226 cells was enhanced obviously. Conclusion Cyclopamine significantly reduced Shh protein expression in five lung cancer cell lines. A significant reducation of Shh protein by Shh-siRNA increased susceptibility of lung cancer cells to radiation and disrupted cyclopamine-mediated radiosenstivity. The present findings demonstrated for the first time that cyclopamine selectively increases radiosenstivity of lung cancer cells, the alteration of Shh expression could be one of the mechanisms involved in cyclopamine-mediated radiosenstivity.