| Severe hepatitis is still a worldwide challenge to physicians. Despite a combination of internal medical treatment, the mortality of severe hepatitis is poor. Liver transplantation remains the only effective therapeutic modality for chronic patients in end-stage, but it is limited by short of the donor organs. Artificial liver support system (ALSS) is a new method for the treatment of severe hepatitis. With the development of technique in isolation of hepatocytes, culture of hepatocytes in high density and bioreactor technique, there are lots of studies focused on the hybrid bioartificial liver support system (HBLSS) which combined with non-bioartificial liver and bioartificial liver. Logically, the hybrid bioartificial liver support system has more powerful function of removing the metabolic products and toxic material in the severe hepatitis patients. Meanwhile, the HBLSS has much more developed function of the biologic synthesis, transformation and detoxification. In this study, we researched on the methods of porcine hepatocyte isolation in high quality and culture, studied the function and morphology of the cell; established a severe hepatitis plasma model to test the efficacy of bioartificial liver support system; construction a vovel hybrid bioartificial liver support system and research its clinical application, test its efficacy and safety; and studied the biological safety of porcine endogenous retro virus (PERV) transmission in HBLSS treatment.1. High quality isolation of pig hepatocytes and clutureWe isolated the porcine hepatocytes by modified two step collagenase perfusion method, and inoculated them into the cultural flask for 1~7 days. Then we observed the viability of the cells, studied the function of the glucose synthesis and diazepam2003transformation, test the albumin & LDH concentration in the supernatant, observed the structure of hepatocytes by converted contrast microscope and electric microscope. The results showed that the total volume of the hepatocytes was 1.5?.4xl010/liver, the viability of the cells was 92?% determined by trypan blue stain. The viability decreased gradually after the in vitro culture. The ALT, AST and LDH concentrations were decreased significantly (P<0.01) after 1~7 days in vitro culture, but the synthesis of Alb and GloB were increased significantly meanwhile. The supernatant glucose concentration was increased gradually in the l~8h culture. The glucose synthetic function was most powerful in the hepatocytes cultured for 1 day, and was stable in the cells cultured for 3 days, 5 days and 7 days. The diazepam transformation function was strongest in cells cultured for 3 days, then diminished gradually. Observed under electric microscope, there was tight juncture and polarity formation between hepatocytes after 1 day culture. It was showed the best morphology and function of the cells cultured for 2~3 day. So we recommend the hepatocytes cultured 2? days for clinical treatment.2. In vitro study of bioartificial liver with a severe hepatitis plasma model The exchanged plasma of the severe hepatitis patients was used as the plasma model. 5?09pig hepatocytes (the viability was above 90%) were cultured in the outer lumen of TECA-I hollow fiber bioreactor for 4~6h after isolation. The bioreactor membrane is mixed cellulose fiber with the diameter of 200nm and molecular weight cut-off of l00kD. The 2000ml exchanged plasma was circulating in the inner lumen of the bioreactor for 3 hours by the driving of pump. After the circulation, the total2003bilirubin concentration was decreased obviously (P<0.05), and other indexes were no statistical changes. There were no obvious changes of the ions concentrations of potassium, natrium, chlochride and calcium. The viability of the hepatocytes was decreased from 92.3?.4% to 73.6?0.3% after 3 hr circulation. It indicated that detoxification should be taken before biological artificial liver treatment. The exchanged plasma of the severe hepatitis patients was a good severe hepatitis model for in...
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