本文選題：mTOR信號通路 + 肝星狀細胞　； 參考：《上海交通大學》2014年碩士論文

【摘要】：第一部分：mTOR信號通路在肝硬化門脈高壓早期演進中活化狀態(tài)及其意義 目的：探討mTOR信號通路在肝硬化門靜脈高壓癥早期演進中的活化狀態(tài)及其意義。 方法：實驗采用膽道結扎離斷制備大鼠早期肝硬化門靜脈高壓癥模型。雄性SD大鼠隨機分為假手術組和模型組。通過組織病理學、形態(tài)學和血流動力學評估肝纖維化、炎癥、和門靜脈壓力。采用RT-PCR和Westernblot法分別測定肝纖維化標志物PC-α1、α-SMA、TGF-β1及PDGF基因的轉錄和mTOR信號通路標志物P70S6K和S6及其磷酸化的P70S6K（P-P70S6K）和P-S6的表達。 結果：大鼠膽道結扎離斷3周后，門脈壓力顯著增高，脾臟顯著增大，而肝內纖維間隔和再生結節(jié)尚未形成，穩(wěn)定構建早期肝硬化門脈高壓大鼠模型；模型組大鼠肝內P-P70S6K（P0.05）和P-S6（P0.05）表達量明顯增高，而總蛋白P70S6K和S6無顯著差異；HSCs和膽管細胞活化增殖顯著，肝臟促纖維化基因PC-α1（P0.05）、α-SMA（P0.05）、TGF-β1（P0.05）及PDGF（P0.05）明顯上調，間質膠原合成增加。 結論：大鼠膽道結扎離斷3周可穩(wěn)定構建早期肝硬化門脈高壓大鼠模型；mTOR信號通路主要以磷酸化功能狀態(tài)參與肝硬化門靜脈高壓癥的形成，阻斷該通路可能成為肝硬化門靜脈高壓癥治療的新靶向。 第二部分：早期肝硬化門脈高壓中雷帕霉素阻斷mTOR信號通路的干預研究 目的：研究雷帕霉素阻斷mTOR信號通路在早期肝硬化門靜脈高壓癥中的治療作用，，并探討其可能機制。 方法：雄性SD大鼠隨機分為假手術組，模型組和模型干預組，采用膽道結扎離斷制備早期肝硬化門靜脈高壓癥模型，模型干預組于術后1周給予雷帕霉素（2mg/kg/d）腹腔注射2周。通過組織病理學、形態(tài)學和血流動力學評估肝纖維化和門靜脈壓力。采用RT-PCR測定肝纖維化標志物基因PC-α1、α-SMA、TGF-β1及PDGF的轉錄；免疫組化檢測α-SMA和增殖性抗原Ki67的表達；Western blot檢測α-SMA、mTOR信號通路標志物P70S6K和S6及其磷酸化的P70S6K（P-P70S6K）和P-S6的表達。 結果：模型組大鼠肝內膽管細胞和α-SMA陽性細胞活化增殖顯著（P0.05），肝脾明顯增大，門靜脈壓力也顯著增高；肝臟促纖維化基因明顯上調（P0.05），蛋白α-SMA（P0.05）、Ki67（P0.05）、P-P70S6K（P0.05）和P-S6（P0.05）的表達量明顯增高；而雷帕霉素通過阻斷mTOR顯著抑制蛋白α-SMA、Ki67、P-P70S6K和P-S6的表達，并改善了肝功能、肝纖維化、門靜脈壓力及脾腫大（P0.05）。 結論：mTOR信號通路是肝硬化門靜脈高壓癥早期演進中重要調控機制，阻斷該通路可能成為早期肝硬化門靜脈高壓癥治療的新靶向。
[Abstract]:Part one: activation of the MTOR signaling pathway in the early progression of portal hypertension in cirrhotic patients objective: to investigate the activation of the mTOR signaling pathway in the early progression of portal hypertension in cirrhotic patients and its significance. Methods: the early cirrhotic portal hypertension model was established by bile duct ligation and dissociation in rats. Male SD rats were randomly divided into sham operation group and model group. Hepatic fibrosis, inflammation, and portal vein pressure were assessed by histopathology, morphology and hemodynamics. The expression of PC- 偽 1, 偽 -SMA-TGF- 尾 1 and PDGF gene, P70S6K, S6 and phosphorylated P70S6K, P-P70S6K) and P-S6 were detected by RT-PCR and Western blot, respectively. Results: after 3 weeks of bile duct ligation and dissection, portal pressure and spleen were significantly increased, while intrahepatic fibrous septum and regenerative nodule had not been formed, so the early cirrhotic portal hypertension model was established stably. In the model group, the expression of P-P70S6KP0.05) and P-S6P0.05) were significantly increased, while the total protein P70S6K and S6 were not significantly different between HSCs and bile duct cells. The hepatic fibrosis gene PC- 偽 1, 偽 -SMAP0.05TGF- 尾 1P0.05) and PDGFP0.05) were up-regulated and the synthesis of interstitial collagen was increased. Conclusion: the early cirrhotic portal hypertension rat model can be stably constructed by bile duct ligation and disconnection for 3 weeks. The signal pathway of mTOR is mainly involved in the formation of portal hypertension in cirrhotic patients with portal hypertension by phosphorylation. Blocking this pathway may be a new target for the treatment of cirrhotic portal hypertension. Part two: intervention study of rapamycin blocking mTOR signal pathway in early cirrhotic portal hypertension objective: to study the therapeutic effect of rapamycin blocking mTOR signal pathway in early cirrhotic portal hypertension. The possible mechanism is also discussed. Methods: male Sprague-Dawley rats were randomly divided into sham operation group, model group and model intervention group. The early cirrhotic portal hypertension model was induced by bile duct ligation and dissociation. The model intervention group was given rapamycin 2 mg / kg / d intraperitoneally for 2 weeks after operation. Hepatic fibrosis and portal vein pressure were evaluated by histopathology, morphology and hemodynamics. The transcription of PC- 偽 1, 偽 -SMA-TGF- 尾 1 and PDGF were detected by RT-PCR, and the expression of 偽 -SMA and proliferative antigen Ki67 were detected by immunohistochemistry. The expression of P70S6K and S6 and phosphorylated P70S6KP-P70S6K) and P-S6 were detected by Western blot. Results: in the model group, the activation and proliferation of intrahepatic bile duct cells and 偽 -SMA positive cells were significantly increased, the liver and spleen were significantly increased, the portal vein pressure was also significantly increased, and the expression of 偽 -SMA-P0.05, 偽 -SMA-P0.05, 偽 -SMA-P0.05, P-P70S6KP0.05 and P-S6P0.05) were significantly increased in the model group. Rapamycin significantly inhibited the expression of protein 偽 -SMA-Ki67, P-P70S6K and P-S6, and improved liver function, liver fibrosis, portal vein pressure and splenomegaly by blocking mTOR. Conclusion the 1: M TOR signaling pathway is an important regulatory mechanism in the early progression of cirrhotic portal hypertension, and blocking the pathway may be a new target for the treatment of early cirrhosis portal hypertension.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575.2

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