摘要
目的进一步探讨PI3K/Akt信号转导途径在药物放射增敏中作用的分子机制。方法体外培养HeLa细胞,多西紫杉醇(docetaxel)和顺铂(cisplatin)单独及分别联合PI3K抑制剂LY294002作用24 h,X线6 Gy剂量照射;Western blot检测Akt、磷酸化Akt(pAkt)、Bad、磷酸化Bad(pBad)蛋白的表达变化;RT-PCR检测Bad、Ku70、Ku80 mR-NA的表达变化;中性彗星电泳检测不同处理组细胞DNA的损伤。结果①docetaxel+LY294002联合照射组、cispla-tin+LY294002联合照射组Bad mRNA表达增高,Ku70 mRNA表达减低,Ku80 mRNA表达无明显变化;②docetaxel+LY294002联合照射组、cisplatin+LY294002联合照射组Bad蛋白表达增高,pAkt、pBad蛋白表达减低,Akt蛋白表达无明显变化;③docetaxel+LY294002联合照射组、cisplatin+LY294002联合照射组细胞彗星电泳尾距明显长于单纯药物增敏照射组。结论①docetaxel和cisplatin药物增敏照射能够明显活化PI3 K/Akt信号转导途径;②抑制PI3 K/Akt信号转导途径能够抑制Ku7 0表达,减少细胞DNA损伤后的再修复,提高促凋亡因子Bad的表达,促进细胞的凋亡。
Objective To further explore the biologic mechanism of the role of PI3K/Akt pathway in radiosensitization of HeLa cells.Methods The HeLa cells were cultured in vitro.Using the IC20 of cisplatin and docetaxel in HeLa or combined with LY294002,the cells were radiated by X-ray.The protein expression of pAkt,Akt,Bad and pBad was detected by Western blot,and the mRNA expression of Bad,Ku70 and Ku80 by RT-PCR.The DNA damages were detected by neutro-comet electrophoresis.Results ①The expression level of Bad mRNA in LY294002＋docetaxel/cisplatin group was higher,and that of Ku-70 mRNA was lower,but there was no significant change in Ku-70 mRNA expression.②The expression level of Bad protein in LY294002＋docetaxel/cisplatin group was higher,and that of pBad and pAkt protein was lower,but there was no significant changes in the Akt protein expression.③The comet electrophoresis tail distance in docetaxel＋cisplatin＋LY294002 group was longer than docetaxel＋cisplatin group.Conclusion The radiosensitization of docetaxel and cisplatin could activate the PI3K/Akt pathway.Inhibiting PI3K/Akt pathway may increase radiosensitization of HeLa cells by inhibiting Ku70 to suppress the DNA recovery,and enhancing the expression of Bad to promote cell apoptosis.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2009年第5期636-640,644,共6页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
教育部新教师基金(No.200804871034)
湖北省自然科学基金(No.2008cdb133)资助项目
