胆管增生和血管新生在胆管缺血性损伤中的作用及机制

发布时间：2018-01-04 10:51

本文关键词：胆管增生和血管新生在胆管缺血性损伤中的作用及机制　出处：《南京医科大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分四氯化碳皮下注射诱导肝硬化大鼠模型目的：建立大鼠肝硬化模型,为大鼠胆管缺血性损伤研究做前期准备。方法：56只SD雄性大鼠随机分为两组,肝硬化造模组50只,对照组6只。50%四氯化碳橄榄油皮下注射诱导大鼠肝硬化模型,每周两次,剂量0.2 mL/100g,连续12周；对照组6只,橄榄油皮下注射,每周两次,剂量0.2 mL/100 g,连续12周。在第4周、8周及12周分别处死2只肝硬化造模组大鼠,监测肝硬化模型形成情况。结果：四氯化碳诱导4周后,大鼠肝脏汇管周围出现肝脏脂肪变性,诱导8周后肝脏脂肪变性向中心静脉区继续进展,汇管区纤维组织增生明显。四氯化碳皮下注射12周的大鼠成功建立了肝硬化模型,肉眼见明显结节性肝硬化特征,光学显微镜下见特征性的假小叶形成。造模组两只大鼠死亡,对照组无大鼠死亡。肝硬化成模率82%。结论：150%四氯化碳橄榄油皮下注射诱导大鼠肝硬化模型安全有效,成模率高。2.该模型制备方法简单,适合大批量的肝硬化模型制备。第二部分胆管增生和血管新生在胆管缺血性损伤中的作用及机制目的：通过对正常大鼠及肝硬化大鼠胆管缺血前、缺血后胆管密度、微血管密度的研究,探讨胆管增生和血管新生在胆管缺血性损伤过程中是否发挥了保护性作用及其可能机制。方法：64只大鼠分成2组(n=32)：①正常大鼠胆管缺血组；②肝硬化大鼠胆管缺血组。每组又随机分Oh、6h、3d和14d四个时间点亚组(n=8)。肝硬化大鼠模型由前期实验的50%四氯化碳橄榄油皮下注射12w制备。分离肝脏周围韧带,结扎肝动脉及肝外肝外胆管周围血管丛(peribiliary plexus,PBP),使肝内胆管完全失去动脉血供,制备胆管缺血模型。静脉血离心取上清,检测血清总胆红素(total bilirubin, TBIL),直接胆红素(direct bilirubin, DBIL),谷草转氨酶(aspartate transaminase,AST)和谷丙转氨酶(alanine aminotransferase, ALT),观察肝脏胆汁瘤形成情况,肝脏组织切片HE染色、Masson染色,采用胆管损伤评分评估胆管缺血损伤程度。肝脏组织切片分别行细胞角蛋白19(cytokeratin 19,CK19)、CD34以及Ki67免疫组织化学染色。CD34评估微血管密度及血管新生情况,CK19及Ki67用来量化胆管密度及胆管细胞增殖情况。应用SPSS19.0统计学软件进行数据处理。结果：总计发现3例肉眼可见的肝内胆汁瘤,均出现在正常大鼠胆管缺血14d组中。正常大鼠血清TBIL水平在胆管缺血后6h、DBIL水平在缺血后3d分别出现显著性升高(P0.01),并在缺血14d达到最高峰(P0.01)。肝硬化大鼠血清TBIL和DBIL水平在胆管缺血6h出现显著性升高(P0.01),并在缺血3d显著下降(P0.01),缺血14d均恢复到缺血前水平(P0.05)。肝硬化大鼠胆管缺血后血清TBIL和DBIL峰值也分别显著低于正常大鼠胆管缺血后的峰值(P0.01)。胆管损伤评分显示肝硬化大鼠及正常大鼠均在胆管缺血6h胆管损伤评分最高(P0.01),随着缺血时间的延长,胆管损伤评分逐渐下降,肝硬化组胆管缺血14d恢复到缺血前水平(P0.05)。正常大鼠组胆管缺血6h、肝硬化大鼠胆管缺血3d均出现了胆管密度显著性增加(P0.01),两组大鼠在缺血6h微血管密度均出现了显著增加(P0.01)。两组胆管细胞增殖活性均在胆管缺血6h出现显著性增强(P0.01),14天降到缺血前水平(P0.05)。在胆管缺血前,缺血后6h、3d和14d,硬化大鼠肝脏微血管密度、胆管密度及胆管增殖活性均分别高于正常大鼠(P0.01)。结论：1.正常大鼠及肝硬化大鼠在胆管缺血后,伴随胆管增生、血管新生,胆管损伤程度逐渐减轻。2.硬化大鼠的肝脏胆管密度和微血管密度高于正常大鼠,其胆管损伤程度轻于正常大鼠。3.胆管增生和血管新生可能分别通过代偿损伤胆管功能和重建胆管血供发挥了对胆管缺血性损伤的保护作用,为胆管缺血性损伤的防治提供了新的靶点和思路。
[Abstract]:The first part by subcutaneous injection of carbon tetrachloride induced liver cirrhosis rat model objective: to establish a rat model of cirrhosis, rat ischemic injury of bile duct preparation. Methods: 56 male SD rats were randomly divided into two groups, liver cirrhosis model group with 50 rats, control group rat liver cirrhosis model induced by 6.50% of four carbon tetrachloride olive oil subcutaneous injection, two times a week, a dose of 0.2 mL/100g, for 12 weeks; the control group 6, subcutaneous injection of olive oil, two times a week, a dose of 0.2 mL/100 g for 12 weeks. In fourth weeks, 8 weeks and 12 weeks were killed 2 rats in liver cirrhosis, liver cirrhosis model formation. The results of monitoring after 4 weeks of CCl4 induced rat liver, portal around hepatic steatosis, after 8 weeks of induction of liver steatosis to the central vein area continues to progress, periportal fibrous tissue hyperplasia obviously. Subcutaneous injection of CCl4 for 12 weeks rats To establish the model of hepatic cirrhosis, the naked eye see obvious nodular cirrhosis characteristics, characteristics of the formation of pseudolobuli seen under a light microscope. The model two rats died, no rat died in control group. Conclusion: 82%. liver cirrhosis model was induced by subcutaneous injection of 150% carbon tetrachloride olive oil is safe and effective in rat liver cirrhosis model, model high rate.2. this model has the advantages of simple preparation method, preparation of liver cirrhosis model suitable for large quantities. The purpose and mechanism of the second part of the bile duct hyperplasia and angiogenesis in bile duct ischemic injury of bile duct ischemia through normal and cirrhotic rats before and after ischemia of bile duct density, microvessel density, investigate bile duct hyperplasia and angiogenesis whether in the bile duct ischemic injury plays a protective role and its possible mechanism. Methods: 64 rats were divided into 2 groups (n=32): normal rat bile duct ischemia group; the liver cirrhosis Bile duct ischemia rats. Each group was randomly divided into Oh, 6h, 3D and 14d four time point subgroups (n=8). The rat models of liver cirrhosis by experimental subcutaneous injection of 12W of 50% carbon tetrachloride olive oil preparation. Separation of the liver ligament, hepatic artery ligation and extrahepatic bile duct (peripheral vascular plexus peribiliary plexus, PBP), the complete loss of intrahepatic arterial blood supply, preparation of bile duct ischemia model. Venous blood was then centrifuged, serum total bilirubin (total, bilirubin, TBIL), direct bilirubin (direct, bilirubin, DBIL), aspartate aminotransferase (aspartate transaminase, AST) and alanine aminotransferase (alanine aminotransferase, ALT), to observe hepatic biloma, liver tissue HE staining, Masson staining, the bile duct injury score evaluation of biliary ischemic injury. Liver tissue sections were examined cell keratin 19 (cytokeratin 19, CK19, CD34 and Ki6) 7 immunohistochemical staining.CD34 evaluation of microvessel density and angiogenesis, CK19 and Ki67 were used to quantify the bile duct and bile duct proliferation density. The application of SPSS19.0 statistical software for data processing. Results: a total of 3 cases of visible intrahepatic tumor, were found in the normal rat bile duct ischemia group 14d TBIL level. The normal rat serum in the bile duct after ischemia 6h, DBIL level at 3D after ischemia were significantly increased (P0.01), and reached the peak in 14d ischemia (P0.01). Liver cirrhosis rats serum TBIL and DBIL levels are significantly increased in the bile duct ischemia 6h (P0.01), and significantly decreased in ischemia (3D P0.01), ischemia 14d were restored to the level before ischemia (P0.05). The bile duct ischemia in cirrhotic rats serum TBIL and DBIL were also significantly lower than the peak peak value of normal rats after bile duct ischemia (P0.01). The bile duct injury score showed liver cirrhosis Rats and normal rats were injured in the bile duct ischemia 6h bile duct (P0.01), the highest score with the time of ischemia and bile duct injury score decreased, liver cirrhosis group 14d to restore bile duct ischemia before ischemia (P0.05). The level of 6h in normal rat bile duct ischemia group, bile duct ischemia 3D in cirrhotic rats showed a significant density of bile duct increased (P0.01), two rats in ischemia 6h microvessel density were significantly increased (P0.01). Two groups of bile duct cell proliferation activity were found in the bile duct ischemia 6h increased significantly (P0.01), 14 days down to the level before ischemia (P0.05). The bile duct before ischemia, 6h after ischemia. 3D and 14d, the liver cirrhosis rat density and microvessel density, bile duct proliferation of bile duct were higher than normal rats (P0.01). Conclusion: 1. normal and cirrhotic rats after ischemia in the bile duct, biliary duct hyperplasia, angiogenesis, gradually reduce the degree of bile duct injury.2. The density of rat liver bile duct and microvessel density was higher than that of normal rats, the bile duct injury in normal rat.3. bile duct proliferation and angiogenesis may play a protective role for ischemic injury of bile duct injury and bile duct through the compensatory function reconstruction of bile duct blood respectively, provides new ideas and target for the prevention and treatment of bile duct ischemic injury.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575.2

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