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

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.

FADD and TRADD expression and apoptosis in primary hepatocellular carcinoma

AIM To investigate the clinical features ofFADD and TRADD expressions in primaryhepatocellular carcinoma(HCC)and todetermine their relationship with hepaticapoptosis.METHODS FADD and TRADD expressions weredetected by immunohistochemistry and hepaticapoptosis were determined by in situ end-labeling(ISEL).RESULTS Ten(25.6%)cases of HCC weredetected to express FADD protein.The positiverate in HCC is lower than that in non-cancerousadjacent liver tissues(62.5%)(P【0.05).Inthose of grade Ⅰ-Ⅱ,8(38.1%)cases wereFADD positive,while only 2/18(11.1%)casesof grade Ⅲ-Ⅳ had detectable FADD protein(P【0.05).No relationship was found betweenFADD expression and other clinical features,such as gender,age,tumor size,differentiationor metastasis.ISEL positive cells can be seen inall cases of HCC.The hepatic apoptosis wasassociated with FADD expression as moreapoptotic cells were detected in those caseswhich had moderately to strongly positiveFADD,as compared with negative or weakpositive FADD cases(P【0.05).No relationshipwas found between FADD expression and hepaticapoptosis in non-cancerous adjacent livertissues.Fifteen of 39(38.5%)cases of HCCwere found positive for TRADD protein,andsimilar positive rate(37.5%)in non-cancerousadjacent liver tissues(P】0.05).The expressionof TRADD is correlated with HCC differentiation, as only 22.2% of moderately to highlydifferentiated HCC showed positive TRADDprotein,while as high as 52.4% of poorlydifferentiated HCC had TRADD(P【0.05),Norelationship was found between TRADDexpression and gender,age,tumor size or gradeor metastasis,although 42.9% of HCC of gradeⅠ/Ⅱ showed positive TRADD which wasslightly higher than that of grade Ⅲ/Ⅳ(33.3%,P】0.05).Hepatic apoptosis was not related toTRADD expression in HCC or non-cancerousadjacent liver tissues.CONCLUSION Loss of FADD expression playsan important role in HCC carcinogenesis,andexpression of TRADD also contributes to HCCdevelopment.The cell apoptosis in HCC isassociated with FADD expression.However,theexpression of TRADD does not correlate wellwith hepatic apoptosis in HCC.

Influence of splanchnic vascular infusion on the content of endotoxins in plasma and the translocation of intestinal bacteria in rats with acute hemorrhage necrosis pancreatitis

INTRODUCTIONThe main reason for the death of the patient with acutehemorrhage necrosis pancreatitis(AHNP)is pancreaticinfection and multi-organ failure caused by endotoxemiaand intestinal bacterial translocation.However,thepathogenesis of endotoxemia and intestinal

Kupffer cell and apoptosis in experimental HCC

INTRODUCTION Our previous study has proved that Kupffer cellsmay have an inhibitory effect on the process ofhepatocarcinogenesis,however,their inhibitorymechanism needs exploring deeply.We performed acomparative study on the expression of PCNA,Bax,P53 and apoptosis of liver cancer cells usingimmunohistochemical technology and terminaldeoxynucleotidyl transferase (TdT)-mediateddUTP-digoxigenin nick end labeling(TUNEL)

The study of emotional presentation after stroke

Abstract: 30 stroke cases were studied in field of their emotional facts. linear related statistics showed the negative relation between the age, disease onset MMSE and ADL with SDS, SSRI drugs and Psychothcrapy should bc useful for prohibiting sever functional retardation.