AFP-shrRNA Combined Mitomycin Affect Huh7Hepatoma Cell

Part OneAFP-shRNA vector amplification,identification and Cultivating human hepatocellular carcinoma cell line Huh7Objective:To amplify,identificate vector of AFP-shRNA and cultivate Huh7cell line of human hepatocellular carcinoma in vitro.Methods:Culture of bacteria.Use axygen Kit to obtain the plasmid vector.The OD values Can be gotten by spectrophotometer.Electrophoresis test the integrity of the plasmid.Hepatocellular carcinoma cells were cultivated in culture bottles and digested by Trypsin to passage generation then Cryopreservation.It can be validated by RT-PCR after Transfection experiments. The PCR amplification products was verified by sequencing.Results:OD260/280of amplified vector was1.91,the electrophoresis results had clear bands. Huh7cell line grew well. There are gene expression by RT-PCR detection and sequencing PCR products tested correct.Conclusion:Successfully obtain the vector with AFP-shRNA Huh7cell is a subset hepatoma cell line which has powerful activity and rapid metabolism.Transfection experiments is successful. Part TwoStudy of AFP-shRNA combined Mitomycin affect Huh7hepatoma cellObjective:Study of AFP-shRNA combined Mitomycin affect Huh7hepatoma cell Methods:Detect the ALT, AST, and AFP after48hours.Detect cell proliferation by CCK-8. Detect apoptosis by flow cytometry,Results:The concentration of ALT,AST,AFP in each transfection dosing group, P <0.05, has significant difference; Compared in each pairwised group, the combined group and afp group, the Mitomycin group, blank group, P <0.05,has significant difference; The afp group compared with the Mitomycin group, P>0.05, has no significant difference. Apoptosis rate of each group, P <0.05, has significant difference; combined group compared with the the afp group and Mitomycin group, blank group, P <0.05, has significant difference; the afp group compared with the Mitomycin group, P>0.05, has no significant difference; Cell proliferation test of each group, P <0.05, has significant difference; Compared the combined group to afp group and blank group, P <0.05, has significant difference; afp group and Mitomycin group P>0.05has no significant difference.Conclusion:1AFP-shRNA can inhibit AFPmRNAexpress,promote apoptosis of hepatoma cells.2AFP-shRNA combined Mitomycin treatment of hepatocellular carcinoma cells can promote liver cancer cell apoptosis and inhibit proliferation of hepatoma cells.