Study Of AFP-shRNA Combined Granzyme B Gene Affect Huh7Hepatoma Cell And Implanted Tumor Of Nude Mice Subcutaneously

Part1Establishment of nude mice hepatocarcinoma model model implanted subcutaneouslyObjective:to establish stable subcutaneous hepatocarcinoma modelMethods:1.5×106cells were implanted into43nude mice individually. After formation tumor, we identify its stability by body weight, gross anatomy, tumor growth, reverse transcription-polymerase chain reaction(RT-PCR), hematoxylin eosin and immunohistochemisty.Results:form tumor ratio of naked mice was97.7%, tumor growth were very rapid, body weight of tumor group is less than control group, expression of alpha-Fetoproteins(AFP) gene and protein was strong positive, hepatocarcinoma cells division were obviously.Conclusion:to establish nude mice implanted subcutaneously hepatocarcinoma model successfully, successful rate was97%. Part2AFP-shRNA-HIV,Granzyme B-LV183lentiviral vector amplification, screening, identificationObjective:to amplify,screen,identificate lentiviral vector of AFP-shRNA-HIV, Granzyme B-LV183 Methods:Lentiviral vector which carrying target gene were amplified and Huh7hepatoma cells were transfected. Calculate the transfection efficiency by fluorescence microscopy and relative gene expression of the transfected groups by RT-PCR, Calculate the Silencing efficiency of target gene based on2'△△ct method. Elect the interference effect was above70%of the Lentiviral vector from the three plasmids by RT-PCR screening method. Compared AFP protein expression differences by Western Bloting. In addition, Established stable Huh7-GrB hepatoma cells by the G418selection, and validated by RT-PCR and Western Bloting. The two PCR amplification products were verified by sequencing.Results:OD260/280of amplified Lentiviral vector was1.86, the electrophoresis results had clear bands. AFP-shRNA-HIV3transfection efficiency is68%,gene silencing efficiency is76.8%, AFP protein expression was significantly decreased. After the Huh7hepatoma cell has been transfected and selected by G418, it has GrB gene expression by RT-PCR. And it had protein expression by Western Bloting detection, and sequencing of PCR products were right.Conclusions:1Successfully elect the interference efficiency of70%of the Lentiviral vector with shRNA-of AFP.2Successfully elect the Huh7-Granzyme B hepatoma Part3Study of AFP-shRNA combined granzyme B gene affect Huh7hepatoma cellObjective:To research the effect of AFP-shRNA combined granzyme B gene on Huh7hepatoma cell and its possible mechanism.Methods:Based on part two, AFP-shRNA lentiviral vector was transfected to Huh7hepatoma cell, detect the ALT, AST, and AFP after48hours. Detect apoptosis by flow cytometry, detect cell proliferation by CCK-8. detect AFP mRNA, Bcl2mRNA, Caspases3mRNA relative expression levels by RT-PCR. these levels are compared with the other groups.Results:ALT,AST,AFP of supernatant of all groups had significant difference(P<0.05), AST,ALT,AFP was significant different in ASG group and KB, AS,GrB,KZLgroup(P<0.05), there were not statistical different between AS group and GrB group,there were not statistical different between in KB group and KZL group.There were statistical different in cell apoptosis of all groups,cell apoptosis was different in ASG group and KB,KZL,AS,GrB,KZL group(P<0.05). Cell proliferation was different in ASG group and KB. AS,GrB,KZL group(P<0.05), there were not statistical different between KB group and KZL group,there were statistical different AS group and GrB gruop (P<0.05). Relative expression of AFPmRNA,Bcl2mRNA,Caspases3mRNA was significant different in all groups,there were significant different in ASG group and KB,AS,GrB,KZL groups, there were not statistical different between in AS group and GrB group, there were statistical different between ASgroup and other groups.Conclusions:1,AFP-shRNA combined granzyme B gene treatment of hepatocellular carcinoma cells can promote liver cancer cell apoptosis and inhibit proliferation of hepatoma cells.2,AFP-shRNA combined granzyme B gene treatment can inhibit AFPmRNA, Bcl2mRNA express,promote Caspases3mRNA expression and promote apoptosis of hepatoma cells.The possible mechanism of AFP-shRNA combined granzyme B treatment lead to apoptosis of hepatoma cells may have two aspects:1,inhibit AFP, Bcl2-expression and inhibit hepatoma cell growth;2,Caspases3is activatied after the GrB enter the hepatocellular carcinoma cells, with the role of the shAFP, it can inhibit AFP expression.Thus inhibit resistant generation of liver cancer cells, and enhance the role of GrB, resulting in apoptosis of hepatoma cells. Part4AFP-shRNA combined granzyme B gene affect implanted tumor of nude mice subcutaneouslyObjective:Study of effects and possible mechanisms about AFP-shRNA combined granzyme B treatment on liver cancer.Methods:Based on the establishment of the nude mice model of liver cancer by part one and the amplification of plasmid by part two, The Nude Mice which had been implanted were randomly divided into five groups, The KBW group, the ASW group, GrBW Group, ASGW group and KZLW group, after the tumor growth in nude mice, injection of gene therapy everyday, tumor diameter and short diameter were measured at3,6,9,15days, and calculate the tumor volume, draw the tumor volume-time growth curve. Mice were killed after15days treatment. Detect of AFP and Bcl2protein expression by immunohistochemistry. Detect AFPmRNA, Bcl2mRNA and Caspases3mRNA relative expression level by RT-PCR. Detect AFP, Bcl2, caspase-3protein expression by Westren Bloting detection.Results:tumor volume in ASGW group is smaller than other four groups, P<0.5. Levels of AFP protein, Bcl2protein, AFPmRNA and Bcl2mRNA in ASGW group were significantly lower than the other four groups, while Caspases3mRNA expression was significantly higher than the other four groups. Levels of Bcl2protein expression in ASGW Group was significantly lower than the other four groups, while Caspases3protein was significantly higher than the other four groups by The Western Bloting.Conclusions:AFP-shRNA combined granzyme B gene treatment can significantly inhibit tumor growth. AFP-shRNA combined granzyme treatment can inhibit AFPmRNA and Bcl2mRNA express, which inhibit the AFP protein, Bcl2protein, while promote Caspases3mRNA expression, thus promote apoptosis of hepatoma cells.The mechanism of AFP-shRNA combined granzyme B lead to apoptosis of hepatoma cells may have two aspects:1,inhibit AFP, Bcl2-expression and inhibition of hepatoma cell growth;2,Caspases3is activatied after the GrB enter the hepatocellular carcinoma cells, with the role of the AFP-shRNA, it can inhibit AFP expression,thus inhibit resistant generation of liver cancer cells, and enhance the role of GrB, promote apoptosis of hepatoma cells.