Study On Establishing Transgenic Tupaias And Mouse With HBx, K-ras Gene And A Cell Model Of Efficient Infection Of Primary Tupaia Hepatocytes With Human Hepatitis B Virus

Hepatocellular carcinoma(HCC)is one of the most common cancers in the world.HBV-associated carcinogenesis can be seen as a multi-factorial progress that includes both direct and indirect mechanisms that might act synergistically.Hepatitis B virus(HBV)is a major etiologic agent of HCC and more popular in developing countries.It has been estimated that about 53%of HCC cases in the world and 90%in China are related to HBV.In recent studies, it was revealed that HBsAg carriers have a 25-37 times increased risk of developing HCC as compared to non-infected people.So it is of great importance that studies on HBV are carried on.Due to deficiency of multiplying system and valuable animals and cell models,little was known about the early mechanism of initiation of infection which limited the study of molecular mechanism in the pathogenesis of hepatitis virus-associated HCC.Our study was performed to investigate the possibility of using the way of testis gene transfer to establish transgenic tupaias and mice with hepatitis B virus X gene and mutated ras gene to study the mechanism of HBV in hepatocarcinogenesis in vivo.Another way,we selected tupaia as the provider of primary hepatocytes to establish the HBV infection model in vitro with further study to explore the function of hepatitis B virus X protein(HBx)and inhibitory effect of IFN-a on HBV replication cycle. PartⅠCloning and Sequence Analysis of HBx Gene and Its Eucaryocytic Expression Vector ConstructionObjective To further investigate the relations between HBx gene and liver cancer by HBx gene cloning and the construction of pcDNA3.1(+)-HBx eucaryocytic expression vector.Methods HBx gene was cloned by PCR from HBxAg plasmid constructed from serum of hepatitis B virus positive patients (serotype adrq~+)of Guangxi Province.HBx gene was directivity inserted into pcDNA3.1(+)plasmid by double enzyme cut.HBx gene sequence analysis and its phylogenetic tree were constructed by DNAStar5.01 and vector NTI Suite 8.0 software.Results HBx gene and pcDNA3.1(+)-HBx eucaryocytic expression vector were successfully cloned and constructed.It is a mutant type of wild genotype C of HBx gene.The new gene was accepted and logged in GeneBank, and accession number is AY839630.There are 97.8%homogeneity and 2.2% divergence compared with HBx gene wild-type C by alignment and phylogenetic tree analysis.Conclusion We had cloned HBx gene mutant genotype C from serum of HBV positive patients.The construction of pcDNA3.1(+)-HBx eucaryocytic expression vector will lay down the foundation to further establish the HBx gene transgenic tupaias and investigate the role of HBx gene in pathogenesis of HBV- associated liver cancer.PartⅡConstruction of pEGFP-N1-K-ras Expression Vector and Its Expression in Different liver Cell LinesObjective To construct pEGFP-N1 eukaryotic expression vector by using enhanced green fluorescence protein as reporter gene and transfecting two different liver cell lines.Methods The mutated K-ras gene was amplified with PCR technique and inserted into pEGFP-N1 vector.The human hepatoma cell line(Huh7.5)and chicken hepatoma cell line(LMH)were transfected with the recombinant plasmid by means of liposome.The EGFP protein was viewed directly with fluorescence microscope,and the expression of K-ras-EGFP fused protein was detected by Western blot.Results The recombinant plasmid was formed correctly by the analyses of enzyme restriction and DNA sequencing,and the 3' end of mutatd K-ras gene was fused with EGFP reporter gene.The green fluorescence could be seen in both Huh7.5 and LMH cell lines and the transfection rate is 19%and 53%respectively.The expression of fusion protein could be detected by Western blot.Conclusion The expression vector of recombinant plasmid pEGFP-N1-K-ras is successfully constructed and is effectively expressed after being transfected into Huh7.5 and LMH,which provides support for the study of establishing K-ras transgenic tupaia model and the function of K-ras gene in hepatocellularcarcinogenesis.PartⅢStudy on Establishing Transgenic Animals by Transfection of Spermatogonial CellsObjective A study was performed to establish a favorable condition for tupaias's breeding and investigate the possibility of using direct testis injection to establish transgenic animals with hepatitis B virus X gene and mutated ras gene.Methods The male and female adult tupaias were matched for breeding pairs.Files were made for each animal.Three methods(Formulated milk, passively and actively maternal milk)were used to feed the pups.Then the two recombinant vectors of pcDNA3.1-HBx and pEGFP-N1-K-ras coated with liposome were injected into the testicle tissue of 5 male mice and 14 male tupaias.The treated animals were fertilized with females four weeks later.The tails of 2～4 weeks old first generation mice were cut and liver biopsies were performed on the baby tupaias after one month of their birth.Polymerase chain reaction(PCR)was performed to detect integration of the target gene into the genomic DNA of the offsprings.Results All the male mice and tupaias injected with recombinant plasmids had reproductive ability.35 first generation mice and 33 first generation tupaias were born.3 mice were PCR positive(positive rate: 8.6%).Among them,2 were positive for HBx,1 was positive for both HBx and ras.2 of the 33 baby tupaias were positive for HBx(positive rate:6.1%). Conclusion It is practical for using the spermatogonial cells as the vector to produce the transgenic animals of HBx and ras gene,and could play a role in the study on the mechanism of HBx and ras gene during hepatocarcinogenesis.PartⅣStudy on Two Isolating and Culture Methods of Primary Tupaia Hepatocytes in VitroObjective To explore the isolating methods of tupaia hepatocytes for primary culture.Methods The hepatocytes from adult tupaias and new-born tupaias were isolated by Two-step perfusion and Percoll working solution respectively and were cultured in vitro.The viability was assessed by typan blue exclusion and MTT methods.The morphologic changes of cultured hepatocytes were observed by the phase contract microscope.The cultured hepatocytes were identified by PAS staining.Results The viability of the hepatocytes isolated from adult tupaias was higher than those from new-born tupaias.The hepatocytes from new-born tupaias were of better proliferation ability than those from adult tupaias.Numerous glycogens were detected in the cytoplasm of the cultured cells from both adult and new-born tupaias by PAS staining.Conclusion Both of the methods are suitable for the culture of primary tupaia hepatocytes in vitro.PartⅤStudy on the Infection of Primary Tupaia Hepatocytes with HBV in VitroObjective To provide a better cell model of closely nature infectious state for further research of Hepatitis B virus.Methods Primary tupaia hepatocytes were isolated according to the two-step perfusion method.The hepatocytes were then infected with purified serum from patients with Hepatitis B.DNA or RNA isolated from the hepatocytes was detected with Southern blot or Northern blot.HBsAg in supernatant was detested by ELISA Kit and the expression of HBsAg in cells was detected by Immunohistochemical method.Results cccDNA,ssDNA,pgRNA and sgRNA could be detected by Southern blot and Northern blot and strong signals could be seen from day7 to day14 post-infection.The S/CO value of HBsAg in supernatant decreased from day1 to day5 and then increased after day5.The expression of HBsAg could be detected in cell plasma on day14 and the positive rate was about 10%. Conclusion Primary tupaia hepatocytes are competent for infection with HBV. HBV can stably replicate and express in HBV-infected tupaia hepatocytes.PartⅥFurther Study Using the Cell Model of Efficient Infection of Primary Tupaia Hepatocytes with HBV ChapterⅠHBV with HBx Gene Mutated can Infect Primary Tupaia HepatocytesObjective To access the function of HBx protein in HBV replication. Methods Huh 7.5 cells were transfected with HBV(HBX21)plasmid.HBX21 is an X-minus mutant by introduction of a stop codon at the beginning of the HBx gene.HBV DNA was detected by southern blot and HBx protein was detected by Western blot.Then the supernatant of Huh7.5 collected on day 5 post-transfection was purified by PEG2000 and added to the primary tupaia hepatocytes(PTH).On day 8 post-infection,the PTHs were harvested.DNA was prepared and HBV DNA was detected by Southern blot.Results HBx protein was not expressed and ssDNA was detected in Huh 7.5 cells transfectd with HBV(HBX21)plasmid,cccDNA could be detected by Sourthern blot in PTH infected with supernatant.Conclusion The presence of HBV replicated intermediates suggests HBx gene is not central for the life cycle of HBV in vitro.ChapterⅡEvaluation of Inhibitory Activity of IFN-a on HBV Replication in VitroObjective To evaluate inhibitory activity of IFN-a on HBV replication in vitro,and to illustrate the possible mechanisms for its target on viral replication. Methods IFN-a had been added to the PTH infected with HBV at various concentration for 5 days.Then HBsAg secreted in supernatant was detected by ELISA Kit.The HBV DNA was detected by Sourthern blot.The HBV RNA and MxA were detected by Northern blot.Results MxA could be induced by IFN-a with a concentration-dependant manner.HBsAg,pgRNA and sgRNA could be inhibited by IFN-a and the inhibitory activity showed a concentrationdependant manner,whereas there was no obvious inhibition on cccDNA.Conclusion In PTH infected model,HBV replication was inhibited by IFN-a.