兔肝纖維化模型的建立及自體骨髓干細胞移植治療
[Abstract]:Purpose 1. A preparation method of rabbit liver fibrosis animal model is established. Evaluation criteria. 2. Establishment of an animal model of hepatic fibrosis rabbit autologous bone marrow mesenchymal transition G Methods: To observe the number, migration and distribution of labeled cells in liver of rabbit liver after autologous bone marrow mesenchymal stem cell transplantation, and to detect the change of serum liver function index. and liver Histopathological analysis and evaluation of clinical efficacy. Methods 1. Establishment and evaluation of rabbit liver fibrosis model: 40 ordinary Japanese white rabbits were randomly divided into experimental group and experimental group. In the control group, 40% CCl _ 4 olive oil solution was injected into the abdominal cavity of the experimental group, and the same amount of normal saline was injected into the control group. The dosage was 0. 5 ml/ kg at 8 weeks after the start of the experiment. The liver group was taken from 30, 60 and 90 days after the start of the experiment. The pathological observation of HE staining and the detection of biochemical indexes were carried out in the tissues and serum samples. The culture and labelling of autologous bone marrow mesenchymal cells were carried out: 24 ordinary Japanese rabbits were randomly divided into experimental groups. In the control group, autologous bone marrow mesenchymal cells were transplanted in the experimental group and normal saline was infused in the control group. The experimental group model rabbits were subjected to percutaneous puncture to acquire bone marrow and density gradient centrifugation to separate the mononuclear cells from the bone marrow, the adherent cells were cultured to obtain the mesenchymal stem cells, and the green fluorescent protein (GFP) was used to mark the cells. The number of labeled cells was observed under fluorescence microscope, and the positive rate was 99%. 3. Autobone marrow mesenchymal stem cell transplantation was used for the treatment of hepatic fibrosis model rabbits and efficacy evaluation: the GPP gene was labeled with 3 ~ 10 ~ 6 cells/ ml bone marrow mesenchymal stem cells were implanted into the experimental group via superior mesenteric vein. In animal liver, the serum of rabbit was taken for liver function test at 0, 4, 8 and 12 weeks after transplantation, and the small part of liver tissue was taken as paraffin section, and the number, migration and distribution of transplanted cells were observed under fluorescence microscope. HE staining was performed on small hepatic tissue and hepatic fibrosis was observed. The changes of pathological histology after cell transplantation were analyzed. The statistical analysis showed that the experimental data were expressed in the form of mean square deviation ((?)/ s), and the statistical indexes of liver function of serum were analyzed by SPSS 13.0 software. The results of analysis of variance and LSD method were used for comparison among multiple levels. Inspection The independent sample t test was used for the comparison between the two horizontal groups, and the test value was P 0.05. The results showed that the model rabbit liver group Histopathological changes: The appearance of the liver was dark red in the control group. The pathological section showed that the structure of the hepatic lobule was complete, and the hepatocytes were arranged radially around the central vein. In the experimental group, the liver was dark red at 8 weeks, there were slight changes in the surface of the liver, and the pathological section showed that the liver cells The focal spot and focal necrosis, infiltration of inflammatory cells in the area of remit, showed early fibrosis. The liver was gray brown at 12 weeks, and there were obvious changes in granulation samples. The pathological section showed: The hepatic lobule structure was destroyed, hepatic cable arrangement disorder, liver cell fat degeneration, interstitial fibrous tissue proliferation, inflammatory cell infiltration, and obvious hepatic fibrosis expression. 2. Model rabbit blood biochemical index: As the time of injection CCL4 was prolonged, the ratio of white globulin decreased from 1. 26 to 0. 93, 0. 17, globulin, indirect bilirubin, direct bilirubin increased gradually, glutamic pyruvic transaminase, glutamic acid and ammonia were transferred to ammonia. The enzymes were elevated to 392. 00, 57. 93, 282. 00, 38. 54. 3, respectively, from 36. 80, 7. 66, 23. 40, and 14. 20. Morphological observation: The primary cells showed the characteristic spiral growth of MSCs, the arrangement of the cells was directional, the center cells of the vortex were distributed in multiple layers, the cell boundary was not clear, and the cell morphology Multi-shuttle-shaped. The growth rate of P1-generation cells increased significantly, and the colony-forming method The results showed that the cell morphology tended to be consistent. GFP-labeled cells were green fluorescence, and the positive rate was 99%. The fluorescence cells observed after transplantation were observed in the liver fibrosis tissues. The amount of green fluorescent cells is uniformly distributed and is compatible with the liver tissue, and the fluorescence cell migration range is wide, As time progresses, positive staining gradually increases and gradually extends inside the liver tissue to the liver's edge. 5. Histopathological observation after transplantation: remove 4 weeks after implantation, the control group had hepatic cord disorder, interstitial fibrosis, partially extended into the lobules, inflammatory cell infiltration, and the recovery of liver tissue structure in the treatment group was obvious. the above changes are lighter and degeneration, necrosis, The cells were significantly reduced. After 12 weeks after transplantation, there was little degeneration and necrosis of hepatocytes in the transplantation group, and a small amount of collagen fibers were deposited around the central vein and the junction area. The structure of the hepatic lobule was clear. Compared with the control group, there was no significant difference. 6. The serum indexes after transplantation were changed: the serum TP content after bone marrow stem cell transplantation. Compared with the control group, the content of GLO decreased gradually, the content of GLO in the transplantation group decreased gradually, the content of serum TBIL and DBIL decreased gradually in the transplantation group, and the content of serum TBIL and DBIL in the transplantation group decreased gradually. photograph group Compared with the control group, the difference between the 4th, 8th and 12th week was very significant (P <0.01). (P <0.01). Conclusion 1. Intraperitoneal injection of 40% carbon tetrachloride olive oil solution 1 2 weeks, the rabbit liver fibrosis model was successfully established: the changes of pathological histology and liver function biochemical indexes were in accordance with the criteria of hepatic fibrosis.
【學(xué)位授予單位】:昆明醫(yī)學(xué)院
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2009
【分類號】:R575.2;R-332
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