The Effects Of Hepatocyte Transplantation On Liver Copper Metabolic Disorder Nude Mice

Objective: 1. To establish the model of liver copper metabolic disorder in nude mice by copper overloading diet.Then it is investigated that the effects of hepatocyte transplantation on liver copper metabolic disorder nude mice through hepatocyte transplantation. 2. To explore the possible mechanism of hepatocyte transplantation in the influence of liver copper metabolic metabolism..Methods: 1. Establishing liver copper metabolic disorder nude mice model : nude mice(4weeks, weight 23±2.3g), they lived in the SPF asepsis animal feeding room well and adapted to the environment after 10 days,then were fed forage with 1.0 g/kg copper sulfate, and 0.185% of bluestone in water for four weeks. 28 d later, the 24-hour urine of nude mice was collected by metabolic cage to examine copper value in the urine. At the same time, the model was evaluated by liver function testing and serum copper test. 2. 40 liver copper metabolic disorder nude randomly divided into 2 groups: Hepatocyte transplantation group(group 1) and culture fluidcontrol group(group 2). There were 20 nude mice in each group. 20 normal nude mice were chosen(they were as old as group1 and group 2 but in normal diet)as normal diet control group(group 3). 40 normal nude mice were chosen for hepatocytes donor. Hepatocytes suspension was prepared and transplanted into nude mice.transplantation.3. Experiment tests: 0.1 ml blood of nude mice was tested for liver function(ALT,ALB,AST), serum copper content and ceruloplasm at1 h before transplantation and 3d, 7d, 14 d and 28 d after transplantation. Cytochrome protein P450(CYP450), malonic dialdehyde(MDA), reactive oxygen species(ROS),liver copper and Superoxide Dismutase( SOD) were tested at 7d and 28 d after operation.At 28 d after operation, biopsy was performed for pathologic exam and liver copper. 4. Statistical analysis: Data were presented by mean ± deviation, counted rate and constituent ratio. Statistical significance was set at P<0.05. All statistical analysiswas processed by SPSS19.0 statistical software.Results:1.After molding, model group serum copper was(4.38±0.17μg/ml)and serum copper of normal feeding group was(1.05±0.05μg/ml),the former wa s higher(P<0.05). The urine copper of the model group was(137.6±13.4μg/24h)and the urine copper of normal feeding group was(25.5±6.1μg/24h),the former was higher(P<0.05). Model group liver function detection of ALT, AST values were higher than normal feeding grou(P <0.05), model group ALB value was lower than normal feeding group(P <0.05). Th e establishment of the model was supported.2. Nude mice liver cell suspension were prepared well, the trypan blue staining cell activity was greater than 90%. After operation 28 d,three groups of nude mice were no death. Liver cell group mass of 1 and 3 can be seen after 28 days, cell mass biopsy in HE staining found liver cells grow well.3. Before and after operation, group 1 and 2 at each time point of AST, ALT, serum copper values were significantly higher than group 3, and the level of serum ceruloplasmin and ALB were significantly lower than group 3, there was significant difference(P<0.05). 1h before operation the testing results of AST,ALT, ALB, serum copper and ceruloplasmin between group 1 and group 2had no significant difference(P > 0.05). 3d, 7d, 14 d,and 28 d after operation the AST, ALT, ALB, serum copper, ceruloplasmin test results between group 1 and group 2 were significantly different(P < 0.05). 28 d after operation the group 1 liver copper content(35.97±1.45μg/g) was lower than the group 2 liver copper content(53.30± 3.17μg/g)(P < 0.05).4. The HE staining of liver tissue appeared: 28 d after the operation, liver cells stained with HE of group 1 were disorganized and some cells swelled with a bit diluted and transparent. Group 2 liver cells stained with HE were ballooning degeneration, fatty degeneration.Group 3 liver cells stained with HE centralvein was clear, liver cell cytoplasm and nuclei were clear, no inflammatory cells infiltration.5. 28 d after the operation group 1 CYP450 2E1 level(0.95 ± 0.02 nmol/mg) compared with the group 2 level(2.41±0.06 nmol/mg) was lower(P < 0.05); the level of MDA(4.25± 0.14 nmol/mg) compared with group 2 level(7.72±0.10 nmol/mg) was lower(P < 0.05); the content of ROS(11.28 ± 1.26 nmol/mg) compared with the group 2 level(25.39±2.33 nmol/mg) was lower(P < 0.05); while the content of SOD(285.59±7.11u/mg.prot) compared with the group 2 level(220.42±7.99 u/mg.prot) was higher(P < 0.05).Conclusions:1. Hepatocytes allograft of nude mice can effectively reduce the copper deposition in nude mice of liver copper metabolism disorder. It also can improve the liver function, accelerate the metabolism of copper. 2. The protective mechanism of hepatocytes transplantation may be related to the reducing of lipid peroxidation injury induced by metabolic disorder of copper.