Human Fetal Hepatocytes To HBV Infection And Construction Of HBV Adefovir-resistant RtN236T Mutant And The RFLP Analysis

HBV infection is one of the commonest infections in the world.According to WHO,about 2 billion people has been infected with HBV,and about 5% are chronically infected. Chronic hepatitis B can cause cirrhosis and liver cancer, more than 1 millon people with this infection are estimated to die every year, and 320 thousand people with liver cancer associated with HBV are to die every year.there are about 28 millon people with chronic hepatitis B in our country.Antiviral therapy is now the main method to CHB, the major medicine includings:interferon, lamivudine, adefovir. Interferon alpha therapy is only moderately effective and often limited by dose-dependent side effects. Honkoop et al. have observed phenotypic and genotypic resistance in 4 patients and estimated that the actuarial incidence of resistance would be 39% at 1 year. The results showed that HBV strains present in serum taken after viral recurrence were about 10000 times less sensitive to lamivudine than those in the pretreatment serum Adefovir is an acyclic nucleotide analog of adenosine monophosphate which is phosphorylated to the active metabolite adefovir diphosphate by cellular kinases. Adefovir diphosphate inhibits HBV DNA polymerase (reverse transcriptase) by competing with the natural substrate deoxyadenosine triphosphate and by causing DNA chain termination afterits incorporation into viral DNA.A number of inherited hepatic disorders and liver failures have been treated with orthotopic liver transplantation (OLT). Unfortunately, the number of patients who have benefited from OLT is very limited, because of the shortage of healthy donor livers. Therefore, techniques have been developed to isolate human hepatocytes for transplantation into recipient livers. Studies have been undertaken to improve the methods related to hepatocyte transplantation, which include hepatocyte isolation from donor livers, cell culture propagation of the hepatocytes, hepatocyte preservation, and genetic modification of the hepatocytes to provide liver specific functions. After many years of basic research, these approaches have moved into the clinics, where more than 50 patients have been treated by hepatocyte transplantation either with allogeneic [9>10>n>12>13>14>15] or autologous hepatocytes with or without genetic modification in vitro [16'17'. For this reason, the transplantation of hepatocytes into recipient livers has the potential advantage for providing treatments of inherited liver disorders and liver failure regardless of the etiology f18>19]. It is important to note that the hepatocyte transplantation procedure is much less invasive and less expensive for patients than OLT. With further modifications, we and others believe that hepatocyte transplantation could offer beneficial effects to a number of patients with liver diseases, who are awaiting OLT, as a viable alternative therapy.Professor Yang finished hepatocytes transplantation for a patient through spleen artery in 302 hospital on oct 29,2004.It has been proved that the orperation is very safe without acute rejection.Sources of donor cells: Primary hepatocytes,,Fetal hepatocytes.bone marrow stem cells, hepatic progenitor cells from embryonic or umbilical cord blood stem cells, liver stem/progenitor cells, immortalized hepatocytes and so on.In this article,I discuss the following problem including hepatocyte isolation from fetus, cell culture propagation of the hepatocytes, hepatocyte cold preservation, feteal hepatocytes infected with HBV,construction of HBV adefovir-resistant rtN236T mutant and the restriction fragment length polymorphism analysis. Objectives:1 Providing reliable human fetalhepatocytes for hepatocytes transplantation in clinic use2 Understanding human fetal hepatocytes viability after cold preservation and thawing.3 Oberving the results that different HBV infecting fetal hepatocytes4 establish an easy method for the detection of HBV Adefovir- resistant rtN236T mutantsMethods:1 Isolation and culture of human fetal hepatocytes1.1 fetal livenobstetric department,nanfang hospital1.2 Using two step methods to separate hepatocytes1.3 Culturing fetal human hepatocytes in the culture dishes2 hepatocytes cold presevation and thawing2.1 4 °C preservation of Fresh human fetal hepatocytes2.2 -196°C preservation of Fresh human fetal hepatocytes2.3 Thawing:using normal quick thawing method2 HBV infecting human fetal hepatocytes2.1 Using HBV wild type and HBV YMDD mutation to infect hepatocytes separately and comminglly2.2 Collecting superior liquid and detecting.4 HBV rtN236T mutant was constructed by site-directed mutagenesis.A novelmethod of mismatched PCR in combination with enzyme digestion was used todistinguish the rtN236T mutant from HBV wild type.Results:1 The digestive method by umbilical vein was available on isolating HFH. The totalproduction viability of hepatocytes was94.76%,Human fetal hepatocytes couldculture for 10-12d in vitro.2Under4°C condition, the viability was 83%, 78%> 10%, 65%, OinCk 30> 54,104n 126h separately, under -196°C condition, the viability was 84.17%s 82%>84%in lw> 2w> 2m separately3 Observing the HBsAg> HBV DNA in supernatant4 The HBV rtN236T mutant was proved by sequencing. By using the new method of PCR restriction flagment length polymorph- ism (RFLP),5%-10%HBV rtN236T mutant type can be easily distinguished from HBV wild type.Conclusions:1 Digestive method via umbilical vein should be first selected on isolating HFH.2 Providing reliable cold preservable human fetal hepatocytes for hepatocytes transplantation in clinic use3 HBV wild type infected human fetal hepatocytes more easily than HBV YMDD mutant type4 The novel PCR-RFLP can be applied to monitor HBV Adefovir-resistant rtN236T mutant.