Experimental Study Of Effect On Huh-7Hepatoma Cells In Nude Mice Subcutaneously Implanted Tumor When Using Granzyme B Gene Combines Curcumin Treatment

Objective:Explore granzyme B, curcumin and combines two treatments'effect on human hepatoma Huh-7nude mouse subcutaneous implanted tumor, and the effect on Caspase-3, Bcl-2apoptotic genes with granzyme B gene expression; The role of curcumin on tumor cell apoptosis. Explore the possible mechanism of granzyme B and Curcumin anti-tumor, provide the experimental basis to find new therapeutic targets for primary hepatocellular carcinoma.Methods:Designed one kind of plasmid carring the granzyme B gene, amplificated the plasmids and transfected into nude mice implanted tumor.Select25BALB/c nude mice,18-22g per each, ages4-6weeks.4-6generations on logarithmic phase hepatoma cells, trypsin digested, adjust cell concentration of1×107/ml suspension, take150ul to implant to nude mice. Divided the25mice randomly into5groups, when the tumor increased to5~7mm. For A group is the granzyme B gene transfection treatment group. For group B is the curcumin treatment group. For group C is the granzyme B combines Curcumin treatment group. For group D is the blank plasmid group. For group E is the control group. Group A:GranzymeB transfection reagent intratumoral injection once a day. Group B:Curcumin100ul intratumoral injection once a day. Group C:GranzymeB transfection reagents, Curcumin100ul interval injection once a day. Group D:blank plasmid transfection reagent intratumoral injection once a day. Group E:only injection Opti-MEM serum-free medium100ul once a day. Every five days measures of the weight of nude mice, tumor volume and draw tumor growth curve. After15days mice were killed to measure tumors' weight, tumors'diameter, and routine pathological examination, RT-qPCR tested Granzyme B, Caspase-3, Bcl-2genes' expression.Result:Amplified plasmid purity meet the follow-up test requirements.Nude mice implanted tumor sucussed, after5days subcutaneous palpable nodules. after12days tumor volume reached5-7mm. The injection therapy groups (A, B, C group) tumor growth were significantly slow, Group C tumor growth slower compared with group A,B and has statistically significant. Group D and Group E, both tumor growth fast compared with the three injection treatment groups, the volume increases significantly. Tumor biopsy were shows:Group D and Group E tumor cells are densely packed, has small necrosis area, cell shapes sizes, and a large deeply stained, visible mitotic activity, nuclear condensation is less. A, B, and C tumor cells are necrosis, shows the nucleus is completely dissolved, the cell structure disappears, homogeneous, red dye cell necrosis performance, you can still see apoptotic cells, cytoplasmic concentration, deeply stained nuclei, solid reduction. The most obvious is the group C, cshows that the cells large areas necrosis, vacuolar change. Real-time PCR analysis shows:A, C groups granzyme B, caspase-3gene expression rised compared to group E, Bcl-2gene expression Declined compared to Group E. B, C groups caspase-3gene expression rised compared to group E, Bcl-2gene expression declined compared to group E. Group D, Caspase-3gene nor Bcl-2gene expression compared to group E had no significant differences.