RAD001对胃癌MKN-45细胞生长影响实验探究.docVIP

RAD001对胃癌MKN-45细胞生长影响实验探究 　 作者：蔡红星，朱正秋，朱祖安【摘要】 目的 探讨RAD001对胃癌细胞株MKN-45细胞生长的影响及可能作用机制。方法 体外培养MKN-45细胞，给予RAD001干预后通过细胞计数检测MKN-45细胞数目，流式细胞术检测细胞周期分布，Western-blot检测核糖体40S小亚基S6K蛋白激酶(p70S6K)、真核起始因子4E(eIF-4E) 结合蛋白1(4E-BP1)、S6蛋白表达及磷酸化情况。结果 RAD001作用于MKN-45细胞24 h：RAD001对MKN-45细胞的生长产生抑制作用，抑制作用具有时间依赖性;MKN-45细胞细胞周期发生改变，停滞在G0-G1期细胞比例增加;MKN-45细胞的4E-BP1、p70S6K、S6无明显变化，phosphate-p70S6K、phosphate - S6表达缺失，phosphate 4E-BP1表达下降。结论 RAD001是通过抑制mTOR 下游通路阻断4E-BP1 及p70S6K的磷酸化和蛋白质翻译进而调节细胞周期以发挥抑制MKN-45细胞生长的作用。 【关键词】 胃癌;MKN-45细胞;RAD001; 细胞生长;抑制作用;机制 　　Abstract: Objective To investigate the effect of RAD001 on gastric carcinoma cell line MKN-45 and to explore the possible mechanism. Methods Gastric carcinoma MKN-45 cell line were cultured in RPMI1640 with 10% fetal calf serum and treated by RAD001 (20 nmol/L); afterwards, the cell coulter, flow cytometry, western-blot array were used to detect the growth, cell cycle and protein expression of the tumor cells. Results Following RAD001 treatment, the MKN-45 cells were inhibited in a time-dependence manner. There were changes in the cycle of the MKN-45 cells, with an increase in the cell percentage of G0-G1 phase stagnation. Meanwhile, there were no marked changes in the expressions of eukaryotic translation initiation factors eIF-4E (eIF-4E) and phosphate-4E-BP1, ribosomal protein 40S kinase, and there was loss in the expression of phosphate-p70S6K and phosphate-S6, and the expression of phosphate 4E-BP1 was down-regulated. Conclusion RAD001 can inhibit the growth of gastric carcinoma cell line MKN-45 by the adjustment of cell cycle via inhibition of the downstream pathway of mTOR and the consequent blockage of the phosphorylation and protein translation of p70S6K and 4E-BP1. 　　Key words: gastric carcinoma; MKN-45 cell; RAD001; cell growth; inhibitory effect; mechanism 　　细胞生长和细胞增殖是相互不同但又相关的两个过程,它们在器官的生长、组织的形成及肿瘤的生长中起着重要的作用。mTOR(mammalian target of rapamycin)是雷帕霉素(rapamycin)的靶分子，是一种丝氨酸/苏氨酸蛋白激酶，在感受营养信号、调节细胞生长与增殖中起着关键性的作用[1]。mTOR信号通路调控异常与肿瘤发生密切相关，是肿瘤