结肠癌细胞球高耐药性及其相关蛋白RAD51和BRCA1的表达  

The relationship between high drug-resistance of spheres from colon cancer cells and the expressions of related proteins RAD51 and BRCA1

作　　者：曹先圣[1] 程勇[1] 杨小丁[1] 耿雪静[2] 庞云[1] 

机构地区：[1]重庆医科大学附属第一医院胃肠外科,重庆400016 [2]重庆医科大学附属儿童医院心血管科,重庆400014

出　　处：《肿瘤》2013年第8期676-683,共8页Tumor

基　　金：重庆市自然科学基金资助项目(编号:cstc2012jjA0038)

摘　　要：目的:探讨结肠癌HCT116和SW480细胞球的高耐药性与同源重组中心分子RAD51和人乳腺癌易感基因1(breast cancer susceptibility gene1,BRCA1)表达的关系。方法:通过无血清悬浮培养获得结肠癌HCT116和SW480细胞球,应用FCM法检测HCT116和SW480细胞球及单层细胞中肿瘤干细胞标志物CD133+细胞的含量。不同浓度的5-氟尿嘧啶(5-luorouracil,5-Fu)和奥沙利铂(oxaliplatin,L-OHP)单独或联合干预HCT116和SW480细胞球和单层细胞后,采用CCK-8法检测细胞的增殖并计算各药物的半数抑制浓度(halfinhibitory concentration,IC50),FCM法检测细胞的凋亡率,免疫荧光及蛋白质印迹法检测细胞中RAD51和BRCA1的表达。结果:HCT116和SW480细胞在含生长因子的无血清培养液中形成可稳定传代的肿瘤细胞球,并且细胞球中CD133+细胞含量显著高于单层细胞(P<0.01)。相同浓度药物干预后,肿瘤细胞球与单层细胞相比细胞生存率更高而凋亡率更低,各药物作用HCT116和SW480细胞球的IC50值显著高于单层细胞(P均<0.01)。不同化疗药物干预后,HCT116和SW480细胞球及单层细胞中RAD51和BRCA1的表达水平均上调;相同药物干预后,HCT116和SW480细胞球中RAD51和BRCA1的表达水平均高于单层细胞(P<0.05)。结论:结肠癌HCT116和SW480细胞球耐药性明显高于普通单层细胞,RAD51和BRCA1可能参与了结肠癌细胞球的高耐药性。Objective： To investigate the relationship between drug-resistance of sphere-forming colon cancer cells and the expressions of RAD51 and BRCA1 （breast cancer susceptibility gene 1） proteins.Methods： The spheres from colon cancer HCT116 and SW480 cells were formed using SFM （serum-free medium） culture.The CD133 ＋ populations of colon cancer sphere-forming HCT116 and SW480 cells and the monolayer cells were detected by FCM （flow cytometry）.After the colon cancer spheres and the monolayer cells were treated with different concentrations of 5-Fu （5-fluorouracil） and L-OHP （oxaliplatin） alone and in combination,the proliferation ability was detected by CCK-8 （cell counting kit-8） test,and the IC 50 （half maximal inhibitory concentration） value was calculated.The apoptotic rate was detected by FCM.The expression levels of RAD51 and BRCA1 were detected by immunofluorescence and Western blotting.Results： The stable colon cancer spheres from HCT116 and SW480 cells were formed using SFM culture with growth factors.The CD133 ＋ population proportion of colon cancer sphere-forming HCT116 and SW480 cells was significantly higher as compared with that of the monolayer cells （P〈0.01）.The colon cancer sphere-forming HCT116 and SW480 cells had a higher survival rate and a lower apoptotic rate as compared with those of the monolayer cells after treatment with the same concentration of 5-Fu or L-OHP （P〈0.01）.The IC 50 values of 5-Fu and L-OHP were significantly higher for the colon cancer sphere-forming HCT116 and SW480 cells than those for the monolayer cells （all P〈0.01）.Theexpression levels of RAD51 and BRCA1 proteins were increased in the colon cancer sphere-forming HCT116 and SW480 cells and the monolayer cells after 5-Fu or L-OHP intervention.The expression levels of RAD51 and BRCA1 proteins in the colon cancer sphere-forming cells were higher than those in the monolayer cells after treatment with the same drugs （P〈0.05）.Conclusion： The spheres from colon cancer c

关 键 词：结肠肿瘤 肿瘤干细胞 培养基 无血清 抗药性 肿瘤 DNA修复 肿瘤细胞球 

分 类 号：R735.35[医药卫生—肿瘤]