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Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

World Journal of Gastroenterology 2005年第48期11卷 7585-7590页

作者： Yong-Bo Cheng Ying-Jie Wang Shi-Chang Zhang Jun Liu Zhi Chen Jia-Jia Li Institute of Infectious Diseases Southwest Hospital Third Military Medical University Chongqing 400038 China

AIM： To observe the effects of plasma from patients with severe viral hepatitis （SVHP） on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device.METHODS： hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione （GSH）, catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes.RESULTS： The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum （NCS） was significant in t-test （LDH： t = 24.552, P = 0.001; AST： t = 4.169, P = 0.014）. After exposure to SVHP for 24 h, alterations in GSH status were significant （F = 2.746, P〈0.05） between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h （F = 4.378, ,P〈0.05）. A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHR The membranes of these cells became indistinct and almost all the cells died on d 5.CONCLUSION： Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes, It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.

关键词： Bioartificial liver porcine hepatocytes Cellculture Plasma toxicity

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porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

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World Journal of Gastroenterology 2010年第13期16卷 1660-1664页

作者： Meng, Fan-Ying Chen, Zhi-Shui Han, Meng Hu, Xin-Peng He, Xing-Xing Liu, Yong He, Wen-Tao Huang, Wei Guo, Hui Zhou, Ping Huazhong Univ Sci & Technol Tongji Med Univ Tongji HospKey Lab Organ TransplantMinist Educ Inst Organ TransplantatMinist HlthTongji Med Co Wuhan 430030 Hubei Peoples R China Huazhong Univ Sci & Technol Tongji Med Coll Inst Liver Dis Tongji Hosp Wuhan 430030 Hubei Peoples R China

AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.

关键词： Hepatocyte isolation porcine hepatocytes Reversible immortalization Simian virus 40 large T-antigen

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Isolation and short term cultivation of swine hepatocytes for bioartificial liver support system

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Hepatobiliary & Pancreatic Diseases International 2005年第2期4卷 249-253页

作者： Jun Li, Lan-Juan Li, Hong-Cui Chao, Qian Yang, Xiao-Li Liu, Ji-Fang Sheng, Hai-Ying Yu and Jian-Rong Huang Hangzhou, China Key Laboratory of Infectious Disease, Ministry of Pub- lic Health of China Institute of Infectious Disease, Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China Department of Infectious Diseases The First Affiliated Hospital Zhejiang University School of Medicine Hangzhou 310003 China. lilj@***

BACKGROUND: The demand for the clinical use of hepa- tocytes is increasing. The aim of this study was to develop a method for procurement of high qualitative pig hepatocytes and to evaluate the state of freshly isolated and cultured hepatocytes. METHODS: The domestic extracorporeal circulating perfu- sion apparatus was used to isolate and harvest swine hepato- cytes by the two-step perfusion method with EDTA and collagenase. The viability, function and morphology of the freshly isolated and cultured cells were evaluated and ob- served by the trypan blue exclusion test, biochemical mea- surements, phase contrast microscopy and transmission electron micrography (TEM). RESULTS: The total yield of isolated hepatocytes reached to 1.5(±0.4)×l010 per liver with a viability of 92(±5)%, and the purity of hepatocytes reached to 98% Immediately after isolation, phase-contrast microscope and TEM showed that undamaged hepatocytes appeared bright, translucent and spherical in shape, with a characteristic well-contrasted border. After 24 hours, the concentrations of alanine aminotransferase (ALT), aspartate aminotrans- ferase ( AST ), lactate dehydrogenase ( LDH ), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in the fluid of culture were declined significantly. CONCLUSION: This method of procuring swine hepato- cytes could get high quality cells with active metabolic function.

关键词： bioartificial liver support system porcine hepatocytes cell isolation cell transplantation

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