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

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、4 Gy组和咖啡因+4 Gy组，咖啡因作用后给予4 Gy 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Annexin Ⅴ-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4 Gy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），4 Gy组G₀/G₁期细胞百分率明显升高（P<0.05或P<0.01），咖啡因+4 Gy组协同作用更强。与S-HepG₂-control细胞比较，咖啡因组和4 Gy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。

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

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 293T cells;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＜0.05 or P＜0.01),the effect was more stronger in caffeine+4 Gy group.Compared with S-HepG2-control,the proliferation activities of S-HepG2-Chk-1 ceils in caffeine group and 4 Gy group were decreased,the apoptotic rates were increased (P＜0.05 or P＜0.01),and the percentage of cells at G1/M phage was significantly decreased (P＜0.05 or P＜0.01).Collusion:The hepatoccellular carcinoma stem cells silenced by Chk-1 with positive CD133 are obtained successfully;Caffeine combined with X-ray irradiation can inhibit the cell proliferation and induce the apoptosis,and enhance the G2/M arrest,and both of them have synergistic effects.