氟尿嘧啶联合丝裂霉素通过改变自噬作用抑制胆管癌细胞QBC939衰老并促进凋亡  

Combination of fluorouracil and mitomycin inhibits aging and promotes apoptosis in cholangiocarcinoma cell line QBC939 by changing autophagy

作　　者：胡惠[1] 王宗鼎 李可洲[3] 龚建平[1] 

机构地区：[1]重庆医科大学附属第二医院肝胆外科,重庆400010 [2]重庆市奉节县人民医院普外科,重庆404600 [3]四川大学华西医院肝胆胰外科,成都610041

出　　处：《第三军医大学学报》2013年第18期1915-1920,共6页Journal of Third Military Medical University

基　　金：四川省中医药管理局重点项目(2012-F-028)~~

摘　　要：目的观察氟尿嘧啶(5-Fu)联合丝裂霉素(mitomycin,MMC)对胆管癌细胞QBC939的自噬、衰老、凋亡的影响,并探讨自噬、衰老、凋亡之间的相互关系及相关机制。方法 5-Fu(25、50、100、150、200μg/mL)、MMC(0.25、0.50、1.0、1.5、2.0μg/mL)及两者相应终浓度的混合溶液分别对胆管癌QBC939细胞作用0、24、48 h后,分别用CCK-8法检测细胞增殖并确定后续实验中化疗药物作用时间及联合化疗药物浓度,Annexin V-FITC染色及流式细胞仪检测细胞凋亡,β-半乳糖苷酶染色检测细胞衰老,单丹磺酰尸胺(Monodansylcadaverin,MDC)染色及流式细胞仪检测细胞自噬;用广谱半胱氨酸天冬氨酸蛋白酶(Caspase)抑制剂Z-VAD-FMK阻断凋亡,用3-甲基腺嘌呤(3-MA)阻断自噬并再次用上述方法检测增殖、自噬、衰老及凋亡变化情况。结果 5-Fu联合MMC(终浓度分别为100、1.0μg/mL)作用24 h时,QBC939增殖受到明显抑制且细胞尚未出现大量死亡,利于各项指标的检测,各组差别有统计学意义(F=26.929,P<0.05);自噬抑制剂作用后,自噬减少,增殖抑制,衰老细胞阳性率降低,细胞凋亡率明显增加(P<0.01);凋亡抑制剂作用后,凋亡减少,增殖增强、衰老细胞阳性率增高明显,自噬变化不明显。结论 5-Fu联合MMC能明显抑制胆管癌细胞QBC939增殖及衰老,促进细胞凋亡,这些变化均与联合化疗药物作用后胆管癌细胞自噬作用的变化有关。Objective To determine the effects of fluorouracil and mitomycin on the autophagy, aging, and apoptosis in eholangiocarcinoma cell line QBC939, and investigate the relationship of autophagy, aging, and apoptosis, and the underlying possible mechanisms. Methods Fluorouracil （25 to 200 μg/mL） , mitomycin （0.25 to 2.0 μg/mL） and their corresponding final concentration of the mixed solution were respectively used to treat QBC939 cells for 0, 24 and 48 h, respectively. Cell counting kit-8 （CCK-8） assay was employed to measure cell viability and determine the combined chemotherapy drug concentration by detecting of cell proliferation. Cell apoptosis was detected by flow cytometry with Annexin V-FITC staining, aging by SA-^- Gal staining, and autophagy by flow cytometry with monodansylcadaverin （MDC） staining. Above detections on proliferation, aging, apoptosis and autophagy were carried out again by the same methods after the apoptosis and autophagy were blocked by caspase inhibitor Z-VAD-FMK and 3-methyl adenine （3-MA） respectively. Results QBC939 proliferation was significantly inhibited when combination of fluorouracil and mitomycin （with final concentrations of 100 and 1.0 Ixg/mL respectively） for 24 h, though widespread cell death was not observed. There was significant differences among groups （ F = 26. 929, P 〈 0.05 ） , and compared with the control group, the autophagy, aging and apoptosis were changed significantly （P 〈 0.01 ）. After the treatment of autophagy inhibitor, the level of autophagy was reduced, cell proliferation was inhibited, the amount of senescent cells was decreased, and apoptosis was significantly increased. On the other hand, after the treatment of apoptosis inhibitor, cell apoptosis was suppressed, proliferation was enhanced, cell aging was significantly increased, but no significant change was found in autophagy. Conclusion Combination treatment of fluorouracil and mitomycin significantly not only reduces cell proliferation but also induces apoptosis

关 键 词：胆管上皮癌 氟尿嘧啶 丝裂霉素 自噬 细胞衰老 细胞凋亡 

分 类 号：R735.8[医药卫生—肿瘤] R965[医药卫生—临床医学]