兔肝纤维化模型的建立及自体骨髓干细胞移植治疗
发布时间：2018-10-09 15:37
【摘要】： 目的 1.建立兔肝纤维化动物模型的制作方法和评价标准。 2.建立肝纤维化动物模型兔自体骨髓间充质细胞转GFP基因标记的方法。 3.观察自体骨髓间充质干细胞移植后肝纤维化模型兔肝内标记细胞的数量、迁移和分布规律,并通过检测血清肝功能指标的变化和肝组织病理分析评价临床疗效。 方法 1.兔肝纤维化模型的建立和评价:40只普通级日本大耳白兔,随机分为实验组和对照组,实验组腹腔注射40%CCL_4橄榄油溶液,对照组腹腔注射等量生理盐水。1—3周CCL_4剂量为0.2ml/kg。3—6周CCL4剂量为0.4 ml/kg。6—8周CCL_4剂量为0.5ml/kg。实验开始后30、60、90天分别留取肝组织和血清标本,进行HE染色病理观察和生化指标检测。 2.自体骨髓间充质细胞的培养和标记:取24只普通级模型组日本大耳白兔,随机分为实验组和对照组,实验组移植自体骨髓间充质细胞,对照组输注生理盐水。对实验组模型兔进行髂骨穿刺采集骨髓,密度梯度离心法分离骨髓单个核细胞,贴壁培养获得间充质干细胞,用绿色荧光蛋白(GFP)标记移植细胞,荧光显微镜下观察标记细胞数量,标记阳性率为99%。 3.自体骨髓间充质细胞移植治疗肝纤维化模型兔和疗效评价:将GPP基因标记3×10~6细胞/ml的骨髓间充质细胞经肠系膜上静脉植入实验组模型动物肝内,于移植后第0、4、8、12周定期取家兔血清进行肝功能检测,并取小部分肝组织做成石蜡切片,置于荧光显微镜下观察移植细胞的数量、迁移和分布规律;另对小部分肝组织进行HE染色,观察肝纤维化组织细胞移植后病理组织学变化。 4.统计分析:实验数据以均值±标准差((?)±s)的形式表示,采用SPSS13.0软件对血清肝功统计指标进行重复测量设计方差分析,多个水平组间比较用LSD法检验,两个水平组间比较采用独立样本t检验,检验值为P≤0.05时具有统计学意义。 结果 1.模型兔肝组织病理学改变:对照组兔肝脏外观呈暗红色,病理切片示:肝小叶结构完整,肝细胞围绕中央静脉呈放射状排列。实验组8周时,肝脏呈暗红色,表面有轻微粟粒样改变,病理切片示:肝细胞点状及点灶状坏死,汇管区炎症细胞浸润,呈早期纤维化表现。12周时,肝脏呈灰褐色,有较明显粟粒样改变。病理切片示:肝小叶结构破坏,肝索排列紊乱,肝细胞脂肪变性,间质纤维组织增生,有炎性细胞浸润,为明显肝纤维化表现。 2.模型兔血生化指标:随着注射CCL4时间的延长,白球蛋白比值由原来的1.26±0.90下降到0.93±0.17,球蛋白、间接胆红素、直接胆红素逐渐升高,谷丙转氨酶、谷草转氨酶分别由36.80±7.66、23.40±14.20升高到392.00±57.93、282.00±38.54。 3.骨髓间充质细胞的形态学观察:原代细胞呈MSCs特征性的旋涡状生长,排列有方向性,旋涡中心细胞呈多层分布,细胞界限不清,细胞形态多呈梭形。P1代细胞生长速度明显增快,集落方式生长,旋涡中心细胞呈多层分布,P3代细胞长梭形更加明显,细胞形态趋于一致。GFP标记细胞呈绿色荧光,标记阳性率为99%。 4.移植后荧光细胞观察:肝纤维化组织中可见大量的绿色荧光细胞,密集均匀分布,与肝组织相容,荧光细胞迁移范围广,逐渐向病灶处迁移,在移植后第3天荧光细胞肝小叶中央静脉周围最多,随着时间的推移,阳性染色逐渐增强,并逐步向肝组织内部延伸至肝脏边缘。 5.移植后组织病理学观察:移植后4周,对照组肝索紊乱,间质纤维增生,部分伸入小叶,有炎性细胞浸润,而治疗组肝组织结构恢复明显,上述改变较轻,而且变性、坏死细胞明显减少。移植后12周,移植组兔肝细胞变性坏死极少,中央静脉及汇管区周围有少量胶原纤维沉积,肝小叶结构清晰。与正常对照组比较无明显差异。 6.移植后血清指标改变:骨髓干细胞移植后血清TP含量、ALB含量逐渐升高,与对照组比较无统计学意义(P＞0.05);移植组血清GLO含量逐渐降低,与对照组比较第8周、第12周有显著差异(P＜0.05);移植组血清TBIL、DBIL含量逐渐降低,与对照组比较,第4、8、12周差异极显著(P＜0.01);移植组血清ALT、AST活性逐渐降低,与对照组比较,第4、8、12周差异极显著(P＜0.01)。 结论 1.腹腔注射40%四氯化碳橄榄油溶液12周,成功建立了兔肝纤维化模型:病理组织学改变和肝功能生化指标变化符合为肝纤维化标准。 2.建立了肝纤维化家兔自体骨髓间充质细胞的转GFP基因标记方法,标记阳性率为99%。 3自体骨髓间充质干细胞移植能够部分修复损伤的肝组织,抑制肝纤维化的发展,部分逆转肝纤维化进程,使其向正常结构发展并改善肝功能。
[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.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R575.2;R-332
本文编号：2259859
[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.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R575.2;R-332
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