咖啡因联合电离辐射对沉默Chk-1肝癌干细胞的增殖抑制和凋亡诱导作用  

Inhibitory effect of caffeine combined with ionizing radiation on proliferation of hepatocellular carcinoma stem cells silenced by Chk-1 and its apoptosis-induced effect

作　　者：于雷[1] 王志成[2] 王铁君[1] 

机构地区：[1]吉林大学第二医院放疗科,吉林长春130041 [2]吉林大学公共卫生学院卫生部放射生物学重点实验室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2018年第1期52-57,共6页Journal of Jilin University：Medicine Edition

基　　金：吉林省卫计委科研计划项目资助课题(2015Z009);吉林省教育厅科学技术项目资助课题(JJKH20170828KJ)

摘　　要：目的:利用咖啡因联合X射线照射处理沉默检查点激酶1(Chk-1)的肝癌干细胞,通过检测细胞增殖、周期和凋亡,探讨二者对肝癌干细胞的协同杀伤效应。方法:沉默Chk-1的慢病毒载体转染293T细胞,慢病毒感染肝癌HepG2细胞后,Western blotting检测Chk-1蛋白表达,确定沉默效果,同时设非靶对照,分别命名为HepG2-Chk-1和HepG2-control。利用悬浮培养法获得CD133高表达的肝癌干细胞,命名为S-HepG2-Chk-1和S-HepG2-control,分为对照组、咖啡因组、4Gy组和咖啡因+4Gy组,咖啡因作用后给予4Gy X射线照射,分别利用MTT法检测细胞增殖活性,利用PI单染和AnnexinⅤ-FITC双染流式细胞术检测细胞周期分布和凋亡率。结果:Western blotting法检测,慢病毒感染HepG2细胞后Chk-1蛋白表达明显降低,而非靶对照组则无明显变化,表明成功获得沉默Chk-1的HepG2细胞模型HepG2-Chk-1和非靶对照模型HepG2-control。将HepG2-Chk-1和HepG2-control细胞悬浮培养后,细胞内的CD133蛋白表达水平均升高,表明存在高比例的CD133+细胞,即肝癌干细胞。与对照组比较,咖啡因组和4Gy组细胞增殖活性明显降低(P<0.05或P<0.01),G1/M期细胞百分率和凋亡率明显升高(P<0.05或P<0.01),且咖啡因组S期细胞百分率明显升高(P<0.05),4Gy组G0/G1期细胞百分率明显升高(P<0.05或P<0.01),咖啡因+4Gy组协同作用更强。与S-HepG2-control细胞比较,咖啡因组和4Gy组S-HepG2-Chk-1细胞增殖活性明显降低(P<0.05或P<0.01),细胞凋亡率明显升高(P<0.05或P<0.01),且G1/M期细胞百分率明显降低(P<0.05或P<0.01)。结论:成功获得沉默Chk-1且CD133+的肝癌干细胞,咖啡因和X射线照射均能抑制细胞增殖和诱导凋亡,并增强G2/M期阻滞,且二者具有协同增强作用。Objective:To treat the hepatocellular carcinoma stem cells silenced by Chk-1 with caffeine combined with 4 Gy X-ray irradiaition,and to explore their synergistic killing effects on the hepatocellular carcinoma stem cells by detecting the cell proliferation,cell cycle and apoptosis.Methods:The lentivirus silencing Chk-1 was transfected into the 293 Tcells;after the HepG2 cells were infected by the lentivirus,the Chk-1 protein expression was detected by Western blotting to confirm the silencing efficency,and non-target control was established,and HepG2-Chk-1 and HepG2-control were named.The cells highly expressed CD133 were cultured by suspension culture method,S-HepG2-Chk-1 and S-HepG2-control were named respectively.After the cells were treated by caffeine,they were irradiated by 4 Gy X-ray;the proliferation activity was measured by MTT,and the cell cycle distribution and the apoptotic rate were measured by PI single staining and AnnexinⅤ-FITC double staining using FCM,respectively.For proliferation,cell cycle and apoptosis assays,there were control,caffeine,4 Gy and caffeine+4 Gy groups.Results:The Western blotting results showed that after the HepG2 cells were infected by lentivirus,the Chk-1 protein expression was significantly decreased,but it was not obvious in non-target control group,it demonstrated that the cell models HepG2-Chk-1 and HepG2-control were obtained successfully.After the HepG2-Chk-1 and HepG2-control cells were cultivated by suspension,the CD133 protein expression were increased,it demonstrated that there were high proportion of CD133+cells,they were hepatocellular carcinoma stem cells.Compared with control group,the proliferation activities in caffeine group and 4 Gy group were significantly decreased(P<0.05 or P<0.01),the percentages of cells at G2/M phage and the apoptotic rates were significantly increased(P<0.05 or P<0.01),and the percentage of cells at S phage in caffeine group was significantly increased(P<0.05);the percentage of cells at G0/G1 phage in 4 Gy group was increased(P<0.

关 键 词：咖啡因 X射线 肝肿瘤 肿瘤干细胞 细胞增殖 细胞凋亡 

分 类 号：Q345[生物学—遗传学]