The Experimental Study Of IL-24 And Endostatin Gene Therapy For Hepatocellular Carcionma

Primary hepatocellular carcinoma common malignant tumor in China, is a common hypervascular liver malignancies, with rapid growth, early metastasis, and mortality characteristics, but also a strong dependence of tumor blood vessels._Most patients with PHC treatment, has been an advanced stage, unresectable, other methods of treatment effect is not ideal._In recent years, gene therapy of cancer have yielded encouraging results, following surgery, radiotherapy, chemotherapy, immunotherapy after the fifth model is one of the most potential treatment of malignant. Cancer gene therapy is currently the focus of basic research. Select the appropriate target genes and gene expression vector is the key, at the same time cancer gene therapy research is mainly to find the target gene and explore the multi-gene combination, into in vivo treatment of tumors. Interleukin-24(IL-24) gene can selectively induce apoptosis in various tumor cells and inhibit tumor cell growth, but also has an indirect anti-angiogenesis activity, anti-cancer treatment has good potential, but had no effect on normal cells. Endostatin can directly induce endothelial cell apoptosis, inhibition of vascular endothelial cell proliferation, inhibition of tumor angiogenesis and inhibit tumor growth. Selected as the two genes were pIRES expression vector construct pIRES-IL-24-ES dual-gene eukaryotic expression vectors. We secretion of the two eukaryotic expression vector transfected NIH3T3 fibroblasts with stable expression after selection, will transfer the cultured cells into nude mice abdominal cavity fiber, the importing and Endostatin and IL-24 gene expression at the same time, the joint treatment of PHC, to explore the efficacy of combined gene therapy of cancer.Objective:Construction of eukaryotic gene expression vectors pIRES-IL-24-ES, and to estimate the effect of IL-24, endostatin gene therapy for hepatocellular carcinoma.Method and Result:1.Separation of human fetal liver tissue extract total RNA, using RT-PCR amplified human IL-24 and Endostatin full cDNA sequence, through gene sequencing, the results compared with the GeneBank exactly the same.Use efficient eukaryotic gene expression vectors pIRES, respectively, recombination pIRES-IL-24, pIRES-ES, pIRES-IL-24-ES eukaryotic expression vectors by restriction analysis,PCR and sequencing proved that the gene sequences to be expressed were reported with the GenBank sequences.2.Identified the correct pIRES-IL-24-ES eukaryotic expression vectors, transfection NIH3T3 fibroblasts, RT-PCR detected the expression of IL-24 and Endostatin in NIH3T3 cells in mRNA levels, ELISA detection the expression of IL-24 secretion and Endostatin protein in the supernatant of stable transfection cell lines.3.Transfected pIRES-ES plasmid, pIRES-IL-24 plasmid and pIRES-IL-24-ES plasmid NIH3T3 cell culture supernatant were added to human umbilical vein endothelial cell line ECV304 medium, pIRES-ES group and pIRES-IL-24-ES group could significantly inhibit the growth of ECV304 endothelial cells; and pIRES-IL-24 group did not inhibit endothelial cell growth. The pIRES-IL-24 plasmid and pIRES-IL-24-ES plasmid transfected HepG2 hepatoma cells, can significantly inhibit the growth of HepG2 hepatoma cells, FCM detected a significant increase in apoptosis in HepG2 cells, pIRES-IL-24 group and pIRES-IL-24-ES group in the promotion of HepG2 apoptosis not significantly different.4.The pIRES-IL-24-ES transfected NIH3T3 fibroblasts with stable expression after selection, cultured fibroblast cells into the abdominal cavity of nude mice, RT-PCR demonstrate that transfection of plasmid expression in the abdominal cavity. ELISA detection of peripheral blood and ascites, there are two proteins, the results show that the pIRES-IL-24, pIRES-ES and pIRES-IL-24-ES three kinds of protein expression plasmid also secreted into the blood and ascites fluid, and expression of single-gene transfer group was not statistically significant from others.5. HepG2 liver cancer cells inoculation, the mice were divided into four groups: pIRES-IL-24-ES group, pIRES-IL-24 group, pIRES-ES group, pIRES vector control. Hepatoma cells were inoculated after 0,7 days, the stable transfection of pIRES-IL-24-ES, pIRES-IL-24, pIRES-ES, pIRES plasmid NIH3T3 fibroblast cells were transplanted into the abdominal cavity of nude mice at 3,6,9,12,15 days was measured in tumor size, tumor size of each group to calculate the survival time of nude mice. Statistical analysis of differences in tumor volume among groups; immunohistochemical detection of tumor blood vessel density, to determine the inhibition of blood vessels; by RT-PCR expression of apoptosis-related gene, ELISA detection of the expression of two proteins in peripheral blood, FCM Detection of apoptosis.Conlusion:1.Successfully constructed pIRES-IL-24-ES, pIRES-IL-24 and pIRES-ES three kinds of eukaryotic expression vectors.2. The expressed proteins secreted by each vector was detected in vitro has good biological activity.3 Established HepG2 liver cancer model in nude mice, the above three kinds of expression vectors were introduced into the abdominal cavity of nude mice, the results shows that each group could significantly inhibit tumor growth.The effect of combined treatment of cancer group was significantly better than single gene therapy and control group.