| Objective :1.To explore and establish stable human hepatoma nude mice model with HepG2 cells,and provide a stable and reliable animal model for future study.2.To study the expression of vascular endothelial growth factor with Angiotensin II receptor antagonists in HepG2 hepatic carcinoma model in nude mice,and looking for the possible principles and mechanisms for HCC postoperative recurrence,metastasis and intrahepatic.3.Discussion of regulating expression of angiogenic factors with angiotensin II receptor antagonist on hepatic carcinoma model in nude mice,and providing new ideas for anti-angiogenic therapy in Hepatocellular carcinoma.Methods:Human hepatoma HepG2 cells were cultured in DMEM medium in 5% CO2,37 ?constant temperature incubator,which contained 10% fetal calf serum and 1%anti-double.HepG2 cells were digested with 5% Trypsin digestion enzymesand spread to the third generation for experiments.Suspension of HepG2 cells were subcutaneously injected and intrahepatic injected to establish a stable model of nude mice bearing human HepG2 hepatocellular carcinoma.Candesartan were selected as Angiotensin II receptor antagonists to do this experiment.Successful models were divided into 5 groups: control group(group C,0.9% sodium chloride),low-dose candesartan group(group L,5mg / kg / d),mid-dose candesartan group(group M,10 mg / kg / d),high-dose candesartan group(group H,20 mg / kg / d)and5-fluorouracil group(group 5-FU,25 mg / kg / d).Using gavage method,continuous feeding two weeks,mice were sacrificed on day 15,tumor size were compared,and whether the abdominal and other organs metastasis.Histopathological examinations was necessary for tumor microvessel density(MVD).Stripping tumor tissue,and weighed.Total tumor tissue RNA was extracted by RNA Extraction Kit.Using RT-PCR method to monitor the expression of VEGF m RNA in different groups.Result:1.Subcutaneous injection and intrahepatic injection of HepG2 cells have no difference in tumor formation rate.However,subcutaneous tumor grewfaster and bigger than liver injection in thesame time.The tumor tissue slices of HE indicated that subcutaneous injection had a poorer blood supply and a lower distant metastasis rate than intrahepatic injection.Intrahepatic tumor tissue was measured and calculated,and tumor volume was 1.16 ± 0.41cm3,subcutaneous tumor volume was 2.80 ±0.52cm3,the results were statistically significant(P <0.001).2.Two weeks after candesartan gavage,nude mice body weight in group C and experimental group increased as tumor grew,but lost in 5-FU group.After the mice were sacrificed,group C and group L were different degrees of distant metastases by dissecting,but in group M,group H and 5-FU,tumor tissue grew slowly,and there had no obvious signs of distant metastases.Tumor size in each group were: 2.29±0.11 g,2.09 ± 0.13 g,1.80 ± 0.08 g,1.51 ± 0.08 g,1.46 ± 0.09 g.Compared to group C,tumor weight were significantly reduced in group L,M,H and 5-FU,P<0.01,group H and group 5-FU had no significant difference in comparison,and P>0.05.VEGF m RNA expression were in each group: 0.880 ± 0.009,0.673 ±0.014,0.274±0.013,0.976±0.028.The expression of VEGF m RNA in different doses of candesartan group was significantly decreased compared to group C and5-FU,P <0.001,the difference was statistically significant.And also,candesartan group had significant difference with 5-FU group in expression of VEGF m RNA,P< 0.001.Conclusion:1.Compared with other methods of liver tumor formation,intrahepatic injection of concentrated HepG2 cells is simpler and easierto operate,and withmore advantages in the study of blood supply,distant metastasis and invasion in liver tumor.And also,it can better reflect the individual cancer cell growth process and closer to the development of clinical hepatocellular carcinoma.It's the ideal animal model for anti-angiogenesis therapy in hepatocellular carcinoma.2.Candesartan inhibits the growth of hepatic carcinoma model of the tumor.Different doses of candesartan can decrease the expression of VEGF in tumor tissue,the greater dose,the more obvious influence.Candesartan can competitively inhibit receptor of angiotensin ?,block tumor tissue angiogenesis,but also through the downregulation of VEGF m RNA and other angiogenic factors,thereby inhibiting tumor angiogenesis. |
PDF Full Text Request