摘要
目的研究化疗药物联合自噬抑制剂对结肠癌细胞凋亡以及钙网蛋白(CRT)膜表达的影响。方法以结肠癌细胞HCT116作为研究对象,采用MTT法检测化疗药物奥沙利铂、5-Fu和SN38对细胞的IC50值,流式细胞术检测化疗药物处理前后的细胞凋亡率和CRT膜表达比例的变化,免疫荧光法观察药物处理前后结肠癌细胞CRT表达部位的变化,Western blotting检测药物处理对HCT116细胞自噬的影响,并用流式细胞术检测药物联合自噬抑制剂氯喹(CQ)处理前及处理12h后细胞凋亡率和CRT膜表达比例的变化。结果化疗药物奥沙利铂、5-Fu和SN38处理HCT116细胞12h后,细胞凋亡率和CRT膜表达比例均较对照组有所上升,但差异均无统计学意义。免疫荧光实验提示奥沙利铂处理后,CRT由细胞质转位至细胞膜。Western blotting结果提示3种化疗药物均可诱导HCT116细胞产生自噬,联合CQ后其诱导自噬的作用受到抑制。化疗药物联合CQ处理HCT116细胞后,细胞凋亡率和CRT膜表达比例均较未用药物处理和单纯化疗药物诱导组增高;奥沙利铂联合CQ处理后CRT膜表达比例升高,与药物处理前比较差异有统计学意义(P=0.027)。结论化疗药物奥沙利铂联合自噬抑制剂CQ能使结肠癌HCT116细胞凋亡增多,CRT膜表达比例上升。
Objective To investigate the influence of chemotherapy combined with autophagy inhibitor on apoptosis and calreticulin（CRT） expression on colonic cancer cells. Methods The colon cancer cells HCT116 were taken as the target in the present study. The inhibition rates（IC50） of chemotherapeutics oxaliplatin, 5-Fu and SN-38 were assessed by MTT assay. The changes in CRT expression on the membrane of HCT116 and apoptosis were determined with flow cytometry before and after treatment with chemotherapeutics. CRT location in HCT116 was detected by fluorescent immunoassay before and after treatment with chemotherapeutic agents. The influence on HCT116 autophagy was determined by Western blotting after treatment with these chemotherapeutic agents. The changes in CRT expression on HCT116 membrane and apoptosis were determined with flow cytometry before and after treatment with the chemotherapeutics combined with autophagy inhibitor chloroquine（CQ）. Results The ratio of apoptosis and membrane expression of CRT were elevated 12 hours after treatment with Oxaliplatin, 5-Fu and SN-38, but without statistical significance. Fluorescent immunoassay showed a transposition of CRT from cytoplasm to the membrane after oxaliplatin treatment. Western blotting revealed that oxaliplatin, 5Fu and SN38 induced autophagy of HCT116 cells, and the autophagy was inhibited by the addition of CQ. Flow cytometric analysis indicated that the percentages of annexin V＋ cells and membrane expression of CRT were higher after treatment with the chemotherapy agents combined with CQ. The upregulation of CRT expression on membrane was obviously higher after treatment with oxaliplatin combined with CQ than that before the treatment with these agents（P=0.027）. Conclusion Oxaliplatin combined with CQ may increase the apoptosis rate of HCT116 cells and upregulate CRT expression in the membrane.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2016年第4期273-278,共6页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金青年基金(81201772)
广东省医学科研基金(B2011112)~~
关键词
钙网蛋白
免疫原性
化疗
自噬
calreticulin
immunogenicity
chemotherapy
autophagy
