Inhibition Of HBV Replication And Expression By 10-23 DNAzyme In Nude Mice Bearing HepG2.2.15 Tumor

Hepatitis B virus (HBV) infection is the main reason for acute and chronic hepatitis. Chronic HBV infection can lead to the development of liver cirrhosis and hepatocellular carcinoma. So far, 350 million people get infected with HBV in the world. HBV infection is very common in our country and about 130 million get infected with HBV. Every year, nearly 300,000 patients with HBV died of the complications. Current therapies include interferon and nucleoside analogs, but so far the therapeutic effect is not satisfactory. Therefore, to develop new gene therapeutic drugs which are new, cheap, effective, low side effects, stable and target oriented is the important means for the therapy of HBV.For the gene therapy of viral diseases, the popular field is the antisense technology in recent years, which includes antioligonuclitide, ribozyme and DNAzyme. Ribozyme degrades easily in vivo. Chemical modification can enhance its stability, but reduce its activity, even eliminate its activity. Antioligonucleotide has longer half life time, but it lacks of catalyzing activity and can′t destroy a target gene, just acts as gene blocking. Based on above discussion, their pharmaceutical applications are limited. 10-23DNAzyme can match with specific target mRNA and inactivate target mRNA by cutting it and comprises a catalytic domain composed of 15 deoxynucleotides, flanked by two substrate recognition arms each composed of 8-10 bases that bind to target RNA through Watson-Crick base-pairing.Only higher primates such as human and chimpanzees are sensitive to HBV infection. Establishing available HBV animal model is regarded as a practical task for HBV in vivo study. In first section of our experiment, the aim is to establish an experimental animal model of HBV in nude mice. Methods: Human hepatoma cell line HepG2.2.15 cells (a hepatoma cell line transfected with HBV genome) are maintained in IMDM media and inoculated in 4-6 weeks age male BALB/c pure line nude mice with a total cell number of 1×107 per mouse. We transplanted HepG2.2.15 cells into nude mice subcutaneously or into abdominal cavity of the nude mice. After that the tumor tissue from nude mice bearing tumor are transplanted subcutaneously or into abdominal cavity of another group of nude mice. Result: the transplanted tumor from nude mice inoculated subcutaneously by HepG 2.2.15 or the tumor tissue appears earlier with higher occurrence rate and shorter experimental term compared with the nude mice inoculated into abdominal cavity. HBsAg, HBeAg and HBV DNA are positive in serum from nude mice bearing tumor. In second section of our experiment, we study the inhibition of HBV expression by site sulfur modification 10-23DNAzyme. Methods: similar to the first section, establishing nude mice HBV model inoculated subcutaneously by the tissue from the tumor. The nude mice bearing tumor are divided into 2 groups at random, respectively experimental group and control group. The experimental group is injected site sulfur modification 10-23DNAzyme with 100μg daily into tumor for 5 days and the control group is injected the same volume normal sodium. Result: compared with the control group,HBsAg and HBeAg are inhibited evidently in serum in experimental group.We conclude from the results as follows:The first, after HepG2.2.15 cells inoculate nude mice into abdominal cavity, the tumor has not been found and ascites have lower occurrence rate. The transplanted tumors from nude mice inoculated subcutaneously appear higher and the HBV markers are positive in the serum. So nude mice HBV model inoculated subcutaneously is thought an ideal animal model for HBV in vivo study and for screening and evaluating the antiviral therapeutic drugs.The second, the transplanted tumor from nude mice inoculated subcutaneously of the tumor tissue appear earlier with higher occurrence than HepG2.2.15 cells and the HBV markers are also positive in the serum. The cell culture specification is higher, therefore the nude mice HBV model by inoculating subcutaneously the tumor tissue is a feasible HBV animal model when need a lot of nude mice bearing tumor.The third, site sulfur modification 10-23DNAzyme can inhibit the expression of HBsAg and HBeAg in the serum of nude mice bearing tumor.In brief, we use HepG2.2.15 cells to establish the nude mice HBV model in order to evaluate the inhibition of HBV expression by site sulfur modification 10-23DNAzyme in vivo, so it lays the foundation for the final clinical application.