| Acute liver failure (ALF) is one of the major causes of death among various liver diseases, and there is no satisfying effect for its therapy by medicine and intensive care. Orthotopic liver transplantation was regarded as an effective method to treat the end stage liver diseases, but there are also many practical problems to be solved, such as shortage of donors, expensive costs, and complicated techniques. Hepatocytes transplantation (HCT) is a new therapeutic technique and becoming another effective method to treat all types of hepatic failure. Because it is simpler, cheaper and has fewer side-effects, HCT has the great potential to be used in clinical practice. In this research, several techniques related to HCT were adopted, such as cell seperation and purification, animal model preparation, and then HCT was performed through three routes: portal vein, spleen, and abdomen to deal with acute liver failure on rats. The aim of the research is to define the best route throuth which the normal hepatocyte can be transplanted into the rates with ALF and establish a foundation for the clinical practice of HCT.Materail and Method1. Preparation of ALF model. Sixty of male SD rats were divided into 5 groups randomly, and fasting 12 hours before the experimant. 10% D-gal solution was prepared and injected to each group of rates via abdominal cavity with dosage of 1.2, 1.4, 1.6, 1.8, and 2.0g/kg respectively. Clinical manifestation, numbers of death, hepatic biochemical function and pathological changes were observed and recorded in each group at different time to determine the optimal dosage for preparation of ALF model.2. Cell seperation and purification. Collagenase perfusion was performed on 20 of SD male rats to separate liver cells, and suspension was counted and cell survival rate was caculated by 0.4% hematochlorin staining as usual.3. Hepatocyte transplantation experiments. One hundred and sixty-four of SD male rats with ALF induced by D-gal were randomly divided into 6 groups: portal venous group and its control group (Group I and II), splenic group and its control group (Group III and IV), abdominal group and its control group (Group V and VI). In Group I (30 rats), 1ml of liver cell suspension was injected through portal venous puncture ( approximately 2X107cells), and muscle injection of lOmg/kg of cyclorsporine A was performed at same time. In Group II (26 rats), 1ml of normal saline was injected instead of cell suspension. In Group HI (30 rats), 1ml of cell suspension was injected into splenic cortex, muscle injection of 10 mg/kg of cyclorsporine A was performed, too. In Group IV (24 rats), 1ml of normal saline was injected instead of cell suspension. In Group V (30 rats), 2ml of liver cell suspension was injected into abdominal cavity (approximately 4X 107cells). In Group VI (24 rats), 2ml normal salinewas injected as above.4. Post-transplantation liver function test and morphologic examination. The blood samples from abdominal aorta of each group of experimental rates were drawn on the 1st, 3rd , 5th and 7th day after transplantation. ALT, AST, TBi, ALP were assayed with automatic biochemical analyzer. Liver sample of rats was biopsied and fixed with 10% formaldehyde. HE staining was performed.Results1. According to the experimental observation, the dosage of 1.6mg/kg was determined as best one for inducing ALF of rates.2. With collagenase perfusion, 0.86~2.3 X 108 cells were obtained ( Mean 1.7X 108), and cell survival percentage was 87?5% ( Mean 91.3%).3. The comparison between group I and II shows that no significant differences were found in survival rate of rats during 24 hours (27% vs. 23%) and 1 week (20% vs. 15%) after transplantation (p>0.05). There was no significant differences in ALT, AST, ALP and TBi on the 1st, 3rd, 5th, and 7th day between two groups either, and there were no significant difference in hepatic pathology also.4. The comparison between group III and IV indicates that there was a significant d... |
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