Abstract: Objective: To treat the hepatocellular carcinoma stem cells silenced by Chk-1 withcaffeine 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 cells 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). Conclusion: 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.

Key words: X-ray, cancer stem cell, cell proliferation, caffeine, liver neoplasms, apoptosis