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Objective 1 rabbit animal models of liver fibrosis production methods and evaluation criteria. (2) to establish animal models of liver fibrosis autologous mesenchymal cells transfected with GFP gene marker approach. 3 Observe autologous bone marrow mesenchymal stem cell transplantation rabbit liver fibrosis model number of labeled cells, migration and distribution, and by detecting the changes of serum liver function and liver histopathology analysis and evaluation of clinical efficacy. Method 1. Rabbit liver fibrosis model and assessment: 40 ordinary level Japanese white rabbits were randomly divided into experimental group and control group, the experimental group were injected 40? L 4 olive oil solution in the control group were injected with normal saline. 1-3 weeks CCL 4 dose 0.2ml/kg. 3-6 weeks CCL4 dose 0.4 ml / kg. 6-8 weeks CCL 4 dose 0.5ml/kg. 60, 90 days after the start of the experiment, respectively, and serum specimens from liver tissue samples for pathological observation HE staining and biochemical testing. 2 of autologous bone marrow mesenchymal cells in culture and labeling: Take 24 ordinary level model group Japanese white rabbits were randomly divided into experimental group and control group, the experimental group autologous bone marrow mesenchymal stem cells in the control group infusion of physiological brine. Rabbit model for the experimental group iliac bone marrow biopsy collection, density gradient centrifugation of bone marrow mononuclear cells, adherent cultured mesenchymal stem cells with green fluorescent protein (GFP) marker transplanted cells, labeled cells were observed under a fluorescence microscope quantity, Mark the positive rate was 99%. 3 autologous bone marrow mesenchymal cell transplantation for the treatment of liver fibrosis in rabbits and efficacy evaluation: the GPP gene marker 3 × 10 6 cells / ml of bone marrow-derived mesenchymal cells were implanted into the superior mesenteric vein group model animal liver, 0,4,8,12 weeks after transplantation regularly from rabbit serum liver function tests, and take a small part of the liver tissue made of paraffin sections were observed under a fluorescence microscope, the number of transplanted cells, migration and distribution; another small part of the liver tissue with HE staining of liver fibrosis cell transplantation histopathological changes. 4 Statistical analysis: The experimental data as mean ± standard deviation ((?) ± s) expressed in the form, using SPSS13.0 statistical software on serum liver function indicators repeated measures design analysis of variance, multiple levels of comparison between groups using LSD method test, two horizontal groups were compared using independent sample t test, P ≤ 0.05 when the test is statistically significant. Results 1. Models rabbit liver histopathology: control the appearance of dark red rabbit liver, biopsy showed: lobule structural integrity of liver cells arranged radially around the central vein. Experimental group, eight weeks, the liver dark red surface has a slight miliary change biopsy showed: liver spots and spotty necrosis, periportal inflammatory cell infiltration, showed early fibrosis. At 12 weeks, the liver was brown, there are obvious miliary changed. Biopsy showed: lobule structural damage, hepatic cord disorder, hepatic steatosis, interstitial fibrosis, inflammatory cell infiltration, as significant liver fibrosis. 2 rabbits blood biochemistry: With the injection of CCL4 time, white globulin ratio increased from 1.26 ± 0.90 down to 0.93 ± 0.17, globulin, indirect bilirubin, direct bilirubin gradually increased, alanine aminotransferase , aspartate aminotransferase respectively 36.80 ± 7.66,23.40 ± 14.20 increased to 392.00 ± 57.93,282.00 ± 38.54. 3. mesenchymal cell morphology: The primary characteristic of MSCs cells were swirling growth direction are arranged resistance, multi-vortex center cells were distributed cell boundaries unclear, mostly spindle cell morphology. P1 cells significantly faster growth rate, colony growth mode, multi-vortex center cells were distributed, P3 is more obvious long spindle cells, the cells converge. GFP-labeled cells showed green fluorescence labeled positive rate was 99%. 4 fluorescent cells was observed after transplantation: Liver fibrosis seen a large number of green fluorescent cells, dense uniform distribution, and liver tissue compatibility, migration of a wide range of fluorescent cells gradually migrate to the lesions, 3 days after transplantation fluorescent cells lobular up around the central vein, over time, positive staining increased gradually and progressively extending to the liver tissue to the liver within the edges. 5 Histopathological observation after transplantation: Four weeks after transplantation, control group hepatic cord disorders, interstitial fibrosis, some stretching lobular, inflammatory cell infiltration, and treatment of liver tissue structure recovery significantly, these changes lighter, Moreover degeneration, necrotic cells decreased significantly. 12 weeks after transplantation, transplantation group little rabbit liver cell degeneration and necrosis, central vein and portal area is surrounded by a small amount of collagen deposition, hepatic lobules clear. Compared with normal control group showed no significant difference. 6 post-transplant serum markers changed: Bone marrow stem cell transplantation serum TP concentrations, ALB content increased, compared with the control group was not statistically significant (P> 0.05); transplant serum GLO decreased gradually compared with the control group 8 weeks, 12 weeks a significant difference (P <0.05); transplant serum TBIL, DBIL content decreased, compared with control group, the first 8 and 12 weeks were significantly different (P <0.01); transplant serum ALT, AST activity decreased, compared with control group, the first 8 and 12 weeks were significantly different (P <0.01). Conclusions 1. Intraperitoneal injection of 40% carbon tetrachloride solution of olive oil for 12 weeks successfully established rabbit model of hepatic fibrosis: Histopathological changes and changes in liver function markers of liver fibrosis meet the standard. (2) to establish a rabbit liver fibrosis autologous bone marrow-derived mesenchymal cells transfected GFP gene markers, marker-positive rate was 99%. 3 autologous bone marrow mesenchymal stem cell transplantation to repair the damaged portion of the liver tissue, inhibit the development of liver fibrosis, liver fibrosis partially reversed the process to make it to the normal structure to develop and improve liver function.
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