| Liver failure is one of the major causes of death among various severe liver diseases and end-stage hepatocirrhosis, and there is no satisfying effect for its therapy by medicine and intensive care. Recently, the prognosis for patients with liver failure, or potentially fatal inherited metabolic disorders is greatly improved by orthotopic liver transplantation (OUT). However, wider application of liver transplantation is restricted by shortage of healthy donor livers, high cost and need for life-long immunosuppression. With the development of bioartificial liver support system (BLSS) and hepatocyte transplantation (HTx), some scientists and hepatologists successfuly applicated HTx or hepatocyte based BLSS to treat severe hepatic failure and end-stage metabolic liver diseases. Their data show that primary hepatocyte is important and promising biomaterial of HTx and BLSS.Morphology and function of cultured hepatocyte in vitro could be changed obviously in short-term. To understand these changes and investigate their optimal functional status for HTx and BLSS, rat hepatocytes were isolated and purified by the modified two-step method described by seglen. The yield and viability of isolated hepatocytes was usually assessed-6-immediately using the standard trypan blue exclusion technique. Hepatocytes were inoculated in the cultured medium consisting of Williams'E which was supplemented with insulin, glycogen, transferrin, dexamethasone, nicotinamide , 2% DMSO and 10% fetal bovine serum. The number of viable hepatocytes during cultured period was assessed by MTT assay. The morphological changes of cultured hepatocytes were observed by light microscope, HE staining and SABC immunohistochemistry; the ultrastructural study was carried out by transmission electron microscope; glucose-6-phosphatase(G-6-Pase) activity was examined by cytochemistry method; the concentrations of albumin and urea and leakage of lactate dehydrogenase(LDH) in the supernatant at different cultured days were examined. The average yield of rat hepatocytes was 1.21 ?0.45xl08 cells per rat with an average viability of 93.6 ?2.8%. MTT assay showed that A of primary cultured rat hepatocytes in third day was higher than other days, and it seems to be optimal status to hepatocyte transplantation. Hepatocytes were observed under light microscope, polygon hepatocytes, some of which were binuclear, were aggregated and arranged in group or cord. Rat hepatocytes can survive for 3 weeks in vitro. The morphological structure of rat hepatocytes is normal and clear through HE staining, some of hepatocytes were binuclear. Under transmission electron microscope, a great abundance of organelles, large stacks of rough and smooth endoplasmic reticula, wheel-shaped mitochondria, glycogen rosettes, tight junction, bile canaliculi and particular ultrastructure of hepatocytes were found, cytokeratin 18 was strongly positive expression in isolated rats hepatocyes or primary cultured rat hepatocytes. Under the electron microscope, there were positive expressions of G-6-Pase in-7- rimary cultured rats hepatocytes, and brownish black granules in cytoplasm were observed. Our data showed LDH leakage, albumin synthesis and urea level had fluctuating changes in one week, but the less LDH leakage and the higher albumin synthesis and urea level were found in the third or fourth day.Hepatocyte transplantation has been applited to clinic as an effective therapy. However, it is not clear about the optimal status and time of hepatocytes transplanted. So, rat hepatocytes transplantation was carried out. First, acute hepatic failure (AHF) rats were induced by 2.0g/kg D-galactosamine (D-gal). Hepatocyte transplantation was started 24h after intoxication, and the rats were divided randomly into three groups: rats in group I received intrasplenic injection of 1 X 107 hepatocytes cultured for 3 days; whereas in group II, rats received 1?07 hepatocytes incubated for 8h intrasplenically; In group...
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