| Experimental Study on Hepatic Stem Cell Transplantation in Treatment of Fulminant Hepatic FailureBackground Orthotopic liver transplantation(OLT) is an effective treatment in patients with terminal liver failure or inborn error of liver metabolism,but many patients can't survive until performing OLT due to critical shortage of donor organs and high cost. As an alternative to this, hepatocyte transplantation (HTx) have been proposed for the temporary metabolic support in patients awaiting OLT or spontaneous recovery.lt is demonstrated that HTx could be used to correct metabolic defects and provide metabolic support for injured liver in experimental and clinical study,but the major limitation of this approach is the shortage of donor organs and difficulty in proliferation and differentiation of hepatocytes, The hepatic stem cells(HSCs) are considered to be a bipotential cells which can differentiate into hepatocytes and biliary epithelial cells,transplantation of these cells could provide metabolic support in patients with liver failure,and also stimulate the regeneration of hepatocytes and lead to recorvery.Chinesetranditional medicine had many good experence in treatment of viarous hepaticdesease,and oriental wormwood decoction had proved to be an effective fomula on many kind of hepatitis.The aim of this research were to explore the therapeutic effect of HSC transplantation in treatment of FHF,and study the protective effects of modified oriental wormwood decoction on injuried liver.Part IExperimental Study on Hepatic Stem Cell Transplantation in Treatment of Fulminant Hepatic Failure RatObjective To establish the hepatic stem cells(HSC) proliferation model in adult rat and study the isolation,purification and identification of the rat HSC,and the therapeutic effect of its transplantation in treatment of fulminant hepatic failure(FHF) rat. Methods In establishment of the HSC proliferation model,the male wistar rats were randomly divided into 5 groups administrated with different dosage(5,10,15,20,25 mg/kg B.W.) of 2-acetylaminofluorene(2-AAF) by gavaged, then a standard 2/3 partial hepatectomy(PH) were performed on day 5, different doses were still administrated as preoperation.The biopisies were adopted and examined by optic and electronic microscope at 3,7,11,14 day postoperation for observation of the proliferation of HSC,the HSC were isolated and purified by collagenase(type I) perfusion method and Percoll gradient centrifugation(2000 rpm,20 min), and the male rat HSC proliferation model was established by administration the similar preoperation dosage for 11 days after operation, then the purified cells were cultured in CO? incubator(37 C ,5% CO?), The Albumin,cytokeratin(CK)18,CK19, a -feto protein (AFP) and leucocyte common antigen ( LCA) were identified by immunohistochemical staining; In 24 h after incubation,0.4ml cell suspension (10 /ml) was injected directly into the median lobe of the liver in female rat FHF models induced by D-galactosamine(D-GlaN, 1200mg/kgB.W.),the same volume of culture medium were injected into the liver of control group rats.The serum ALT,AST,TBiL and blood ammonia levels were measured pre and post transplantation at different time points.To identify the male HSCs in female rats, Sex determining region of the Y( Sry) was examined by PCR technique at 1,2,4 weeks after transplantation. Results Pathologic examination of the rat liver revealed that oval cells were induced ideally in rats with administeration of 2-AAF at a dose of 15mg/kg B.W. combined with 2/3 PH on day 5, and then administered 2-AAF daily up to 11 days beginning on the day after operation,HSC appeared in the periportal areas,which was characterized by a oval-shaped nucleus and high nuclear/cytoplasm ratio, these cells were positive for AFP,CK18,CK19 staining,and negative for ALB and LCA; stem cell yield/rat: 5.10+0.40x106 Viability: 94.30+1.89% by Trypan blue excluded test.Thediameter of cells was less than 1/3 the size of the nomal hepatocytes under phase-contrast...
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