| Despite the introduction of numerous new therapeutic approaches, relatively little progress has been made in improving the prognosis of the patient with acute liver failure(ALF).The mortality rate of ALF continues to be unacceptably high with figures range from 60% to 80%.Currently, orthotopic liver transplantation(OLT) is the only curative treatment. Unfortunately, the use of OLT is severely limited by the scarcity of immediately available donors, high cost, high morbidity, the requirement for sophisticated technology and need for life-long immunosuppression.Several bridging techniques have been proposed to sustain patients until an organ becomes available, or liver function recovers naturally, including extracorporeal perfusion of human liver, the use of bioartificial liver(BAL) and hepatocyte transplantation. Hepatocytes transplantation is an attractive alternative to OLT because hepatocytes transplanted can carry out detoxification and synthetic functions normally performed by the liver. In comparsion with OLT, hepatocytes transplantation is technically easy, less invasive, lower morbidity and cost.A key issue of hepatocyte transplantation is how to obtain hepatocyteswith enough number and good function. As a high density culture technique, microcarrier cell culture method has both the advantages of a monolayer culture and a suspension system, and has been used for culture of various anchorage-dependent cells.Here, we describe morphologic as well as metabolic characteristics of rat hepatocytes in microcarrier culture, and also evaluate the effect of intraperitoneal transplantation of microcarrier-attached hepatocytes on acute liver failure in rats induced by D-galactosamine(D-GalN) through comparing biochemical and histologic examination as well as survival rates for the entire experimental period of three groups.The aim of this research is to lay an experimental foundation for the research work of hepatocyte material and provide the experimental basis for the clinical trials of hepatocyte transplantation. The experimental results are as following:1. The modified Seglen two-step method was used to isolate hepatocytes from rats and it resulted in a mean hepatocytes viability of 93.6%+2.8% with a mean hepatocyte yield (1.21 + 0.45) X 108 cells/rat. The morphological characteristics and abilities of albumin and urea synthesis can maintain for above 1 week. The LDH leakage, albumin synthesis and urea level fluctuate in one week, and there is a lesser LDH leakage and higher albumin and urea level on the third day.2. The dosage of D-GalN was chosen as 2.0g/kg in according to the piolt experiment. Twenty-four hours after D-GalN administration, hepatocytes transplantation were performed.3. The survival rate of the rats group 3 which receive the hepatocytes culturedon microcarrier is much higher than that of nude microcarriers group (group 1). Differences of survival rates between group 1 and group 3 were significant on the 5th day and beyond the 5th day of transplantation(P<0.05). Fifty-seven percent of the animals transplanted with microcarrier-attached hepatocytes were alive 14 days after transplantation, Fourty-one percent of the rats transplanted with hepatocyte suspension were alive 14 days after transplantation, whereas only 15.4% of the animals received microcarriers alone were alive at the same time.4. As time goes by, the liver function levels of group 3 decreased and approached normal on 5th day. There were significant differences in ALT, TBiL and AST levels between 1st and 5th day(P<0.05).No decline tendency was observed in the liver function levels of group 1.Serum bilirubin and ALT levels of group 1 peaked on 3rd day. The liver function levels of group 3 on 3rd ,5th day were lower when compared to that of group 1. Significant difference was found in TBiL levels between group 1 and group 3 on 3rd ,5th day(P<0.05). 5 When the abdominal cavity was explored sequentially following intraperitoneal transplantation of microcarrier-attached hepatocytes, most of the ce... |
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