| BackgroudHBV infection is one of the commonest infections in the world. According tothe results from WHO, about 2 billion people have been infected with HBV, andabout 5% are chronically infected. Chronic hepatitis B may develope to hepaticcirrhosis and hepatocellular carcinoma (HCC). More than 1 million people die fromchronic HBV infection per year, and 320,000 people die from HBV associated HCC.In China, there are about 28 millon people infected with HBV with active liverdiseases.25%-40% patients with chronic hepatitis B may die from hepatic cirrhosis andHCC, and the survival ratio of five years is about 15% in patients with hepaticcirrhosis in stage of decompensation. At present, the treatment of severe hepatitiswill take expectant treatment, However, this can not solve fundamental question, andthe sole measure is orthotopic liver transplantation (OLT).A number of patients with inherited hepatic disorders and liver failures havebeen treated with orthotopic liver transplantation (OLT). Unfortunately, the numberof patients who have benefited from OLT is limited, because of the shortage ofhealthy donor livers. The hepatocyte transplantation provide a new thinking andmethod for solving the contradiction, which patients may afford this treatment. The techniques have been developed to isolate human hepatocytes for transplantation intorecipient livers. Studies have been undertaken to improve the methods related tohepatocyte transplantation, which include hepatocyte isolation from donor livers, cellculture propagation of the hepatocytes, hepatocyte preservation, and geneticmodification of the hepatocytes to providing liver specific functions. After manyyears of basic research, these approaches have moved into the clinics, where severalpatients have been treated by hepatocyte transplantation either with allogeneic orautologous hepatocytes with or without genetic modification in vitro. hepatocytes intorecipient livers has the potential advantage for providing treatments of inherited liverdisorders and liver failure regardless of the etiology. It is important to note that thehepatocyte transplantation procedure is much less invasive, less expensive, safer forpatients than OLT. With further modifications, it is believed that hepatocytetransplantation may offer beneficial effects to a number of patients with liver diseases,who are awaiting for OLT, as a variable alternative therapy.There are many sources of donor cells: primary hepatocytes, fetal hepatocytes,bone marrow stem cells, hepatic progenitor cells from embryonic or umbilical cordblood stem cells, liver stem/progenitor cells, immortalized hepatocytes and so on.In this article I discussed the following problem including fetal hepatocyteslong-term culture, fetal hepatocytes infected with the recombined adenovirus vectorcarrying 1.3-fold-overlength genome of HBV DNAObjectives:1. Providing reliable human fetal hepatocytes for hepatocytes transplantation in clinicapplication and research2. Establishing a method for human long-term culture of human fetal hepatocytes3. Observing the basic profiles of fetal hepatocytes infected with the recombinedAdenovirus Vector Carrying 1.3-fold-overlength genome of HBV DNA Methods:1. Isolation and culture of human fetal hepatocytes1.1 Fetal liver from the Obstetric Department, Nanfang Hospital1.2 Using two step methods to separate hepatocytes by umbilical vein perfusion.2. The density of fetal hepatocytes preservation is 5-7×10~6/mL. phase lowertemperature, 4℃60minutes,-20℃30minutes,-80℃18 hours, preservation inliquid nitrogen at last.3. Hepatocytes thawing: using normal quick thawing method, take cold presersationfetal hepatocytes out of liquid nitrogen into the 37℃water, quickly rewarming in1 minute.4. Long-term culture fetal hepatocytes: the fetal hepatocytes were plated inattachment medium containting 5% fetal bovine serum and kept for 4 hours beforechanging the medium to a serum -free Ham's F12 hormonally defined mediumsupplemented, dexamethasone,transferrin,glucagon,insulin,epidermal growth factor(EGF) and growth hormone (GH), changing fresh serum -free difined mediumevery 2 day, hematopoietic cells contained in fetal hepatocytes did not attach andwere eliminated with medium replacements after a few days in culture.5. Recombined adenovirus vector carrying 1.3-fold-over length genome of HBVDNAinfect human fetal heptocytes5.1 Recombined adenovirus vector carrying 1.3-fold-over length genome of HBVDNA: presevation in our laboratory5.2 Fetal hepatocyte'coming from cold presevation fetal hepatocytes5.3 Human fetal hepatocytes were infected with recombined adenovirus vectorcarrying 1.3-fold-over length genome of HBV DNA for 2 hours, washing withmedium for 10 times, collecting supernatants, detecting the express level of HBsAg and HBeAg by Abbott complete automation microparticle enzyme immunoassay,HBV DNA in supernatant by fluorescent quantitation PCR, HBsAg and HBcAg incell by immuohistochemistry.Results:1. The digestive method by umbilical vein was available on isolating human fetalhepatocyte. Under 4℃condition, the viability was 83%,78%,70%,65%,0%, in0,30,54,104,126hour separately, under -196℃condition, the viability was84.17%,82%,84%, in lweek,2week,2month respectively. Observing the fetalhepatocytes by phase contrast biology microscope,cells began to adhere and extendfrom 2-3 hours,which completed about 6 hours, fetal hepatocyetes were full ofculture flasks in Ham s F12 hormonally defined medium after 3 days. Monocyteand dicaryocyte could be observed clearly by phase contrast biology microscope,CD90 and CD34 could be detected by flow-cytometric, specific AFP still could bedetected by Abbott complete automation microparticle enzyme immunoassay,when fetal hepatocytes had been cultured for 10 days,15 days, 30 days and 45days.2. Recombined adenovirus vector carrying 1.3-fold-over length genome of HBV DNAhave infected human fetal heptocytes for 2 hours, washed 10 times, cultured for 7more days, the quantitation of HBsAg, HBeAg and HBV DNA increasingsignificantly,HBsAg and HBcAg were positive stained in fetal hepatocytes byimmunohistochemistry.Conclusion:1. Digestive method via umbilical vein should be first selected on isolating humanfetal hepatocytes2. Providing reliable cold preservable human fetal hepatocytes for hepatocytestransplantation in clinic and scientific research use3. Fetal hepatocytes can be long-term cultured for 2 months in a serum -free Ham' s F12 hormonally defined medium4.HBV can replicate and HBV proteins expressed in fetal hepatocytes... |
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