| Due to its species-specificity, the study on hepatitis B virus (HBV) is always lack of ideal animal model. The mouse model established by transgenic technology has been widely used all over the world, and has played significant roles in the study of the etiopathogenesis of hepatitis B, the treatment of anti-HBV and the evaluation ofanti-HBV drugs, etc.Nowadays, HBV genome has been transmitted stably to F20 generation, and the index of replication and expression of HBV always remains stable. It can be detected by ELISA and fluorescent quantitation PCR, and comparable to the generally acknowledged replicative HBV transgenic mice generated by Guidott. The replicative HBV transgenic mice had provide an ideal animal model for the study on nosogenesis of HBV and anti-HBV drugs. Now this model generated by us has been used by hundreds of scientific research units in our country.This study include: (1) the establishment of replicative 1.3 copy HBV transgenic mice; (2) the study on its biologic characteristics; (3) the application of replicative HBV transgenic mice in the evaluation of anti-HBV drugs; and (4) the generation of non-immune tolerant HBV transgenic mice by intraperitoneally injecting plasmid expressing siRNA specific for HBV.Chapter 1 the establishment of replicative 1.3 copy HBV transgenic mice. In our experiment, 1.3 copy HBV (genotype D) was used as target gene, which was transferred into male pronucleus of fertilized eggs, and then the DNA-injected fertilized eggs was transplanted into mouse oviduct under phantom pregnancy, which developed into off-spring. The off-spring mice were screened by PCR, serum HBsAg and HBeAg were detected by ELISA, and serum HBV DNA was detected by fluorescent quantitation PCR. Result: 1210 fertilized eggs were obtained, 882 was microinjected, 397 was survived, the survival rate was 45%; 337 was transplanted into t4 mice under phantom pregnancy, 9 mice was pregnant and deliver 54 off-spring mice, the successful transplant rate was 16%(54/337). 54 mice was PCR positive, 4 GO mice was serum HBsAg positive, 2 high expressing founders were obtained. 13 serums HBsAg positive was obtained in 61 G1 mice. The expressing amount of serum HBsAg was 80～930μg/. Immunohistochemical technique was used to detect HBsAg and HBcAg in many tissues such as liver, spleen, kidney and small intestine, the results showed that HBsAg and HBcAg are unevenly expressed in liver and kidney.Chapter 2 This chapter make a study of F 14-F20 Balb/C-TgN (HBV 1.3) including: (1) HBV genome replication and expression level and its stability; (2) the property of immunology and pathology; (3) transcriptional level in several tissues; and (4) breeding methods for improve replicative and expressive stability.Firstly, Analysis with serum ELISA and PCR indicated that HBsAg, HBeAg, HBcAg, pre S 1 and HBV DNA were found in the serum of transgenic mice, of which HBsAg level up to 5107.5±4144.SIU/ml but HBeAg lower to 1.944±1.66 s/co and difference existing in different individuals. The characteristic of HBV genome replication and viral particle package and secretion have been analyzed by RT-PCR and transmission electron microscope. Under the transmission electron microscope, HBsAg particle and Dane particle could be found, indicating that HBV genome could be replicated and package into particle to secret into blood. HBV DNA levels in HBV transgenic mice were up to 10~3～10~6copy/ml. This value is an important index for evaluating anti-HBV drugs.Immunohistochemical analysis showed that HBsAg and HBcAg were expressed in hepatocyte, but not all hepatocytes expressing, HBsAg in cytoplasm and HBcAg in nucleus. RT-PCR of different tissues indicated that HBV genome was transcribed in transgenic mice with the highest mRNA level in hepatocyte. The value of mRNA is also an important factor for evaluating drugs. Liver HE staining demonstrated that hepatocellular cytopathic effect was not found in hepatic tissue of HBV transgenic mice. Natural and adaptive immunity was demonstrated normally in transgenic mice except immunotolerance for HBV by flow cytometry and Elispot.In order to keep HBV genome stably transmitted, each of founder mice was passaged independently to stabilize the expression of foreign gene. After stably transmiting of each founder, two founder mice are crossed each other to increase the integrated copy number of genome and to increase the levels of the replication and expression. Analysis with positive rate and HBsAg expression level of F14-F20 showed that HBV genome was stably integrated into mouse gnome and transmitted into next generation, consistent with Mendelian inheritance rule. This kind of breeding approache can increase HBsAg expressing level and positive expression rate.In conclude, the analysis of the bionomics of Balb/C-TgN (HBV1.3) indicate that this mouse strain is very ideal HBV transgenic mouse model. This study have a much better knowledge of transgenic mice which relieving the lack of animal model for hepatitis B and establishing the foundation for hepatitis B research.Chapter 3: The evaluation of many anti-HBV drugs (such as lamivudine, adefovir, interferon-a, HBV curable vaccine and HBsAg mono-antibody) indicated the utility of our HBV transgenic mice.Chapter 4: Non-immune tolerant HBV transgenic mice were primarily established by intraperitoneally injecting plasmid with HBV-specific siRNA.Chapter 5 This chapter summarized and made a prospect on this study. The author have successfully established replicative HBV transgenic mice, HBV genome has been transmitted stably to F20 generation, and had expressed several HBV index, had been applied widely. The model can be used to anti-drug evaluation at the DNA, mRNA, cell and organism level and study on anti-HBV new-drug development. Classic anti-HBV drugs had effect on the model, which reflected that the model can truly simulate many characteristics of human B hepatitis. The positive rate and expression amount of off-spring can be effectively improved by increasing the copies of target gene. In addition, this study also discussed the establishment of non-immune tolerant HBV transgenic mice by intraperitoneally injecting plasmid with HBV-specific siRNA.The author also make a discussion on HBV transgenic mice's role in guiding and checking treatment study of chronic hepatitis B (CHB) by the research achievement and relative literature. The first consideration for cure is anti-HBV drugs combined with immunotherapy. In clinical, effective power is 30% by single anti-HBV drugs, while few patients have a consist reaction to HBV. The trends of CHB treatment is anti-HBV drugs combined with immunologic intervention factor. This kind of immunotherapy involved in specific immunity and inherent immunity. HBV transgenic mice provide an ideal platform for this kind of study for its natural and normal adaptive immunity. Another consideration is maintaining the immune tolerant condition of CHB patient, decreasing hepatic-injury-hepatitis caused by immune response. HBV transgenic mice have not demonstrated any hepatic injury.
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