Establishment and further improvement of D-galactosamine induced acute hepatic failure in Duroc breeding pigs / 中华传染病杂志

Objective To establish and improve the acute hepatic failure model in pigs induced with D-galactosamine (D-gal),and explore the feasibility of evaluating preclinical artificial liver devices.Methods Nineteen Duroc breeding pigs were divided into 4 groups.Fifteen unanesthetic Duroc breeding pigs out of 19 (5 of each group) received intravenously administration of D-gal at a dose of 1.0,1.25 and 1.5 g/kg body weight,respectively.The remaining 4 pigs which received the same volume of 5% dextrose in water served as controls. Clinical data and survival time of pigs were recorded.Blood samples were collected for dynamic testing of plasma ammonia,prothrombin time,liver and renal functions,blood glucose and L-lactate;liver tissues were sampled for pathological examination.The differences between groups were compared using t test and F test.The survival time of pigs was compared by Kaplan-Meier survival analysis and Log Rank test.Results Twelve hours after administration of D-gal,all pigs presented as acute hepatic failure characterized by progressive increases of levels of plasma ammonia,aspartate aminotransferase (AST),total bilirubin (TBil) and L-lactate,the level of blood glucose marked decreased and prothrombin time prolonged (F= 32.33,F=27.817,F=50.097,F=88.382,F=8.211,F=21.227;all P＜0.01);especially in the pigs which received D-gal at a dose of 1.5 g/kg.Except 2 pigs survived for 168 h,the other 3 pigs which received D-gal at a dose of 1.0 g/kg died within 68-84 h,while all pigs which received D-gal at a dose of 1.25 and 1.5 g/kg died within 33-89 h and 23-47 h,respectively.All pigs presented coma before death and liver histopathological examination indicated massive hepatic necrosis with severe hemorrhage.Conclusions D-gal induced acute hepatic failure model in unanesthetic Duroc breeding pig appears potential reversibility and high reproducibility,which has proper therapeutic window.Thus,this model could be applied to evaluate the therapeutic effects and safety of artificial liver devices.

Establishment and further identification of immortalization human hepatocyte lines / 中华传染病杂志

Objective To establish immortalized human hepatocyte lines for studies of bioartificial liver,hepatocyte transplantation,and drug metabolism in vitro.Methods Primary human hepatocytes were isolated by 4-step perfusion technique with collagenase and transfected with recombinant retrovirus containing Simian virus 40 large T antigen(SV40 LT).Subsequently,immortalized human hepatocytes were evaluated by analysis of gene expression and functional characteristics in vitro.Results Two immortalized human hepatocyte lines,HepLi2 and HepLi3,were obtained after primary human hepatocytes being infected by SV40 LT containing recombinant retrovirus for 3-4 weeks.The immortalized human hepatocytes showed classical appearance of hepatocyte observed by phase contrast microscope.The protein expression of SV40 LT in HepLi2 and HepLi3 cells were detected by Western blotting.The mRNA expressions of albumin(Alb),glutathione S-transferase(GST-p),human blood coagulation factor X(HBCF-X)and β-actin in HepLi2 and HepLi3 cells were detected by reverse transcription polymerase chain reaction(RT-PCR),and the mRNA expressions of cytochrome(CY)450 subtypes(CYP3A5,CYP2E1,CYP2C8-19 and CYP3A4)in HepLi2 and HepLi3 cells were also observed by RT-PCR.Levels of alanine transaminase (ALT),lactate dehydrogenase(LDH)and Alb were detected in the supernatant of immortalized human hepatoeyte culture.Conclusions The immortalized human hepatocyte lines have the biological characteristics of primary human hepatocytes and have the CYP450 functions of hepatocytes,which may be heIDful for the studies of bioartificial liver,heoatocvte transplantation and drug metabolism in vitro.

Immortalization of porcine hepatocyte lines by transfection with simian virus 40 large T antigen and human telomerase reverse transcriptase / 中华传染病杂志

Objective To establish normally immortalized porcine hepatocyte lines by ectopic expression of simian virus 40 large T (SV40LT) antigen and the human telomerase reverse transcriptase(hTERT). Methods Primary porcine Hepatoeyte cells were transfeeted with recombinant retrovirus containing SV40LT or hTERT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Immortalized porcine hepatocyte was confirmed by examination. Results The morphological phenotype of the transfected cells was similar to the primary porcine hepatocyte. One clone, HepLP, has been maintained in cultue for half year, and expanded by more than 60 passages. SV40 LT and hTERT could be detected in transfected porcine hepatocyte. Pig albumin mRNA was also detected by RT-PCR. No tumor formation occurred when HepLP cells were injected into Balb/c nude mice. Conclusions The immortalized, nontumorigenic, porcine hepatoeytes maintained the properties of porcine primary hepatocytes such as the albumin secretion. This generation of immortalized porcine hepatocyte may be helpful for bioartifical liver support system, hepatocytes transplantation, drug/toxicological studies, and liver biologic studies.