CD133与磷脂酰肌醇3激酶p85相互作用促进胃癌细胞化疗耐药  

Interaction between CD133 and phosphatidylinositol 3 - kinase p85 promote the chemoresistance capacity of gastric cancer cells

作　　者：宋书铮 姜波健[1] 吴巨钢[1] 倪晓春[1] 王守练[1] 杜亚琼[1] 俞继卫[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院普外科,201999

出　　处：《中华实验外科杂志》2017年第2期188-191,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81101850）;上海市卫生计划生育委员会基金（20134321）

摘　　要：目的构建稳定干扰及过表达CD133的胃癌细胞株，观察CD133分子对胃癌细胞化疗多药耐药能力的影响，并探讨其机制。 方法使用慢病毒在KATOⅢ及MKN45中分别构建稳定干扰及过表达CD133细胞株；Western blot验证干扰及过表达效率。使用细胞计数试剂盒（CCK-8）法检测各组细胞对化疗药物5-氟尿嘧啶（5-Fu）和顺铂（DDP）的敏感性，采用Western blot法检测耐药相关蛋白P-糖蛋白（P-gp）、B细胞淋巴瘤/白血病-2（bcl-2）、B细胞淋巴瘤/白血病-2相关X蛋白（bax）的表达及磷酸化蛋白激酶B（p-Akt）的激活，最后利用免疫沉淀技术检测CD133与p85之间的相互作用情况。 结果与空载体组比较，干扰组或过表达组的CD133蛋白水平明显降低或升高。而相同药物浓度下，5-Fu和DDP对干扰组的抑制率明显高于对照组，其半数抑制浓度分别为[（54.030 0±0.647 9） μmol/L比（133.300 0±4.918 0） μmol/L，P＝0.005和（2.312 0±0.223 6） μmol/L比（24.590 0±2.159 0） μmol/L，P＝0.001]；而对过表达组的抑制率明显低于对照组，其半数抑制浓度分别为[（8.102 0±0.433 1） μmol/L比（2.217 0±0.132 4） μmol/L，P＝0.010和（24.487 0±2.132 0） μmol/L比（11.620 0±1.534 0） μmol/L，P＝0.030]。在稳定干扰CD133表达后，耐药相关因子P-gp、bcl-2和p-Akt的蛋白水平均显著降低，而促凋亡因子bax表达则显著升高；与之相反，过表达CD133后，P-gp、bcl-2和p-Akt的蛋白水平显著升高，而bax则明显降低。而用p85抗体进行免疫沉淀后，过表达组检测到CD133表达。 结论CD133能够显著促进胃癌细胞的化疗耐药能力；而这可能是通过与磷酸肌醇3激酶（PI3K）的亚基p85相互作用进而激活PI3K/Akt信号通路实现的。Objective By building stable CD133 knock- down and overexpression cell lines, we will study its effects on the ability of multiple chemoresistance and the possible relevant mechanism. Methods Firstly, we established KATO Ⅲ - shCD133 and MKN45 - CD133 cell lines, and then tested the interference and overexpression efficiency of CD133 by Western blotting. Secondly, we detected the cells sensibility to 5 - fluorouracil （ 5 - Fu） and cisplatin （DDP） by cell counting kit - 8 （ CCK - 8 ）, and measured the protein level of chemoresistance related factors： P - gp, B cell lymphoma/leukemia - 2 （bcl -2）, B cell lymphoma/leukemia -2 associated X protein （bax） and the activation level of protein ki- nase B （Akt） by Western blotting. Finally, we examined the interaction of CD133 and p85 by immunopre- cipitation. Results By Western blotting, we found the protein level of CD133 of the knock - down or overexpression cell lines were significantly lower or higher than the control group. Under the same drug concentrations, 5 -Fu and DDP showed higher inhibition rate in the CD133 konck- down group than that of the vector group with the half maximal inhibitory concentration （ICs0） value [ （54. 030 0 ± 0. 647 9）μmol/L vs. （133. 300 0 ±4. 918 0） μmol/L, P =0. 005] and [ （2. 312 0 ±0. 223 6） μmol/L vs. （24. 590 0 ±2. 159 0）μmol/L, P=0. 001], while the overexpression group took the opposite sides with the IC50 value [ （8. 102 0 ±0. 433 1 ）μmol/L vs. （2. 217 0 ±0. 132 4）μmol/L, P =0. 010] and [ （24. 487 0 ±2. 132 0） μmol/L vs. （ 11. 620 0 ± 1. 534 0） μmol/L, P = 0. 030]. After stablyknock -down the expression of CD133, the protein levels of chemoresistance related factor： P - gp, bcl -2 and p -Akt were significantly lower, and the promoting apoptosis factor bax was significantly increased; the overexpression group was still the opposite. Conclusion CD133 could obviously increase the chemoresis- tance capacity of gastric cancer ceils, which might be realized

关 键 词：胃癌 CD133 化疗耐药 磷脂酰肌醇3激酶p85 

分 类 号：R735.2[医药卫生—肿瘤]