mTOR信号通路在肝硬化门脉高压早期演进中调控机制的研究

发布时间：2018-06-18 12:08

本文选题：mTOR信号通路 + 肝星状细胞　；参考：《上海交通大学》2014年硕士论文

【摘要】：第一部分：mTOR信号通路在肝硬化门脉高压早期演进中活化状态及其意义目的：探讨mTOR信号通路在肝硬化门静脉高压症早期演进中的活化状态及其意义。方法：实验采用胆道结扎离断制备大鼠早期肝硬化门静脉高压症模型。雄性SD大鼠随机分为假手术组和模型组。通过组织病理学、形态学和血流动力学评估肝纤维化、炎症、和门静脉压力。采用RT-PCR和Westernblot法分别测定肝纤维化标志物PC-α1、α-SMA、TGF-β1及PDGF基因的转录和mTOR信号通路标志物P70S6K和S6及其磷酸化的P70S6K（P-P70S6K）和P-S6的表达。结果：大鼠胆道结扎离断3周后，门脉压力显著增高，脾脏显著增大，而肝内纤维间隔和再生结节尚未形成，稳定构建早期肝硬化门脉高压大鼠模型；模型组大鼠肝内P-P70S6K（P0.05）和P-S6（P0.05）表达量明显增高，而总蛋白P70S6K和S6无显著差异；HSCs和胆管细胞活化增殖显著，肝脏促纤维化基因PC-α1（P0.05）、α-SMA（P0.05）、TGF-β1（P0.05）及PDGF（P0.05）明显上调，间质胶原合成增加。结论：大鼠胆道结扎离断3周可稳定构建早期肝硬化门脉高压大鼠模型；mTOR信号通路主要以磷酸化功能状态参与肝硬化门静脉高压症的形成，阻断该通路可能成为肝硬化门静脉高压症治疗的新靶向。第二部分：早期肝硬化门脉高压中雷帕霉素阻断mTOR信号通路的干预研究目的：研究雷帕霉素阻断mTOR信号通路在早期肝硬化门静脉高压症中的治疗作用，，并探讨其可能机制。方法：雄性SD大鼠随机分为假手术组，模型组和模型干预组，采用胆道结扎离断制备早期肝硬化门静脉高压症模型，模型干预组于术后1周给予雷帕霉素（2mg/kg/d）腹腔注射2周。通过组织病理学、形态学和血流动力学评估肝纤维化和门静脉压力。采用RT-PCR测定肝纤维化标志物基因PC-α1、α-SMA、TGF-β1及PDGF的转录；免疫组化检测α-SMA和增殖性抗原Ki67的表达；Western blot检测α-SMA、mTOR信号通路标志物P70S6K和S6及其磷酸化的P70S6K（P-P70S6K）和P-S6的表达。结果：模型组大鼠肝内胆管细胞和α-SMA阳性细胞活化增殖显著（P0.05），肝脾明显增大，门静脉压力也显著增高；肝脏促纤维化基因明显上调（P0.05），蛋白α-SMA（P0.05）、Ki67（P0.05）、P-P70S6K（P0.05）和P-S6（P0.05）的表达量明显增高；而雷帕霉素通过阻断mTOR显著抑制蛋白α-SMA、Ki67、P-P70S6K和P-S6的表达，并改善了肝功能、肝纤维化、门静脉压力及脾肿大（P0.05）。结论：mTOR信号通路是肝硬化门静脉高压症早期演进中重要调控机制，阻断该通路可能成为早期肝硬化门静脉高压症治疗的新靶向。
[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- 伪 1, 伪 -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- 伪 1, 伪 -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- 伪 1, 伪 -SMA-TGF- 尾 1 and PDGF were detected by RT-PCR, and the expression of 伪 -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 伪 -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 伪 -SMA-P0.05, 伪 -SMA-P0.05, 伪 -SMA-P0.05, P-P70S6KP0.05 and P-S6P0.05) were significantly increased in the model group. Rapamycin significantly inhibited the expression of protein 伪 -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.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2

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