本文關(guān)鍵詞：膽管增生和血管新生在膽管缺血性損傷中的作用及機制　出處：《南京醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 大鼠 四氯化碳 肝硬化 動物模型 血管新生 膽管 膽管細胞 肝硬化 缺血 增殖

【摘要】：第一部分四氯化碳皮下注射誘導(dǎo)肝硬化大鼠模型目的：建立大鼠肝硬化模型,為大鼠膽管缺血性損傷研究做前期準備。方法：56只SD雄性大鼠隨機分為兩組,肝硬化造模組50只,對照組6只。50%四氯化碳橄欖油皮下注射誘導(dǎo)大鼠肝硬化模型,每周兩次,劑量0.2 mL/100g,連續(xù)12周；對照組6只,橄欖油皮下注射,每周兩次,劑量0.2 mL/100 g,連續(xù)12周。在第4周、8周及12周分別處死2只肝硬化造模組大鼠,監(jiān)測肝硬化模型形成情況。結(jié)果：四氯化碳誘導(dǎo)4周后,大鼠肝臟匯管周圍出現(xiàn)肝臟脂肪變性,誘導(dǎo)8周后肝臟脂肪變性向中心靜脈區(qū)繼續(xù)進展,匯管區(qū)纖維組織增生明顯。四氯化碳皮下注射12周的大鼠成功建立了肝硬化模型,肉眼見明顯結(jié)節(jié)性肝硬化特征,光學(xué)顯微鏡下見特征性的假小葉形成。造模組兩只大鼠死亡,對照組無大鼠死亡。肝硬化成模率82%。結(jié)論：150%四氯化碳橄欖油皮下注射誘導(dǎo)大鼠肝硬化模型安全有效,成模率高。2.該模型制備方法簡單,適合大批量的肝硬化模型制備。第二部分膽管增生和血管新生在膽管缺血性損傷中的作用及機制目的：通過對正常大鼠及肝硬化大鼠膽管缺血前、缺血后膽管密度、微血管密度的研究,探討膽管增生和血管新生在膽管缺血性損傷過程中是否發(fā)揮了保護性作用及其可能機制。方法：64只大鼠分成2組(n=32)：①正常大鼠膽管缺血組；②肝硬化大鼠膽管缺血組。每組又隨機分Oh、6h、3d和14d四個時間點亞組(n=8)。肝硬化大鼠模型由前期實驗的50%四氯化碳橄欖油皮下注射12w制備。分離肝臟周圍韌帶,結(jié)扎肝動脈及肝外肝外膽管周圍血管叢(peribiliary plexus,PBP),使肝內(nèi)膽管完全失去動脈血供,制備膽管缺血模型。靜脈血離心取上清,檢測血清總膽紅素(total bilirubin, TBIL),直接膽紅素(direct bilirubin, DBIL),谷草轉(zhuǎn)氨酶(aspartate transaminase,AST)和谷丙轉(zhuǎn)氨酶(alanine aminotransferase, ALT),觀察肝臟膽汁瘤形成情況,肝臟組織切片HE染色、Masson染色,采用膽管損傷評分評估膽管缺血損傷程度。肝臟組織切片分別行細胞角蛋白19(cytokeratin 19,CK19)、CD34以及Ki67免疫組織化學(xué)染色。CD34評估微血管密度及血管新生情況,CK19及Ki67用來量化膽管密度及膽管細胞增殖情況。應(yīng)用SPSS19.0統(tǒng)計學(xué)軟件進行數(shù)據(jù)處理。結(jié)果：總計發(fā)現(xiàn)3例肉眼可見的肝內(nèi)膽汁瘤,均出現(xiàn)在正常大鼠膽管缺血14d組中。正常大鼠血清TBIL水平在膽管缺血后6h、DBIL水平在缺血后3d分別出現(xiàn)顯著性升高(P0.01),并在缺血14d達到最高峰(P0.01)。肝硬化大鼠血清TBIL和DBIL水平在膽管缺血6h出現(xiàn)顯著性升高(P0.01),并在缺血3d顯著下降(P0.01),缺血14d均恢復(fù)到缺血前水平(P0.05)。肝硬化大鼠膽管缺血后血清TBIL和DBIL峰值也分別顯著低于正常大鼠膽管缺血后的峰值(P0.01)。膽管損傷評分顯示肝硬化大鼠及正常大鼠均在膽管缺血6h膽管損傷評分最高(P0.01),隨著缺血時間的延長,膽管損傷評分逐漸下降,肝硬化組膽管缺血14d恢復(fù)到缺血前水平(P0.05)。正常大鼠組膽管缺血6h、肝硬化大鼠膽管缺血3d均出現(xiàn)了膽管密度顯著性增加(P0.01),兩組大鼠在缺血6h微血管密度均出現(xiàn)了顯著增加(P0.01)。兩組膽管細胞增殖活性均在膽管缺血6h出現(xiàn)顯著性增強(P0.01),14天降到缺血前水平(P0.05)。在膽管缺血前,缺血后6h、3d和14d,硬化大鼠肝臟微血管密度、膽管密度及膽管增殖活性均分別高于正常大鼠(P0.01)。結(jié)論：1.正常大鼠及肝硬化大鼠在膽管缺血后,伴隨膽管增生、血管新生,膽管損傷程度逐漸減輕。2.硬化大鼠的肝臟膽管密度和微血管密度高于正常大鼠,其膽管損傷程度輕于正常大鼠。3.膽管增生和血管新生可能分別通過代償損傷膽管功能和重建膽管血供發(fā)揮了對膽管缺血性損傷的保護作用,為膽管缺血性損傷的防治提供了新的靶點和思路。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R575.2

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