肝脏特异性Ggps1敲除通过激活FXR介导胆汁酸排泄改善梗阻性黄疸肝损伤

发布时间：2018-03-05 14:14

本文选题：香叶基香叶基二磷酸合成酶　切入点：梗阻性黄疸　出处：《南京大学》2017年博士论文　论文类型：学位论文

【摘要】：恶性梗阻性黄疸是由于恶性肿瘤生长阻塞肝总管或胆总管,导致胆汁无法排泄至肠道,胆汁的正常肝肠循环被阻断引起。在肝胆外科实践中,恶性梗阻性黄疸通常是肝内胆管癌、胰头癌、远端胆管癌、胆囊癌尤其是肝门部胆管癌患者的伴随症状。在针对肝门部胆管癌的外科治疗中,肝胆外科医生常常需要对肝门部胆管癌患者进行肝脏大部分切除术以达到根治性治疗目的。但目前对于梗阻性黄疸引起的肝损伤是否会抑制肝切除术后肝脏再生能力以及有无其他更有效的方法改善梗阻性黄疸引起的肝损伤未能有很深入的认知。因此本课题拟基于相关动物实验探讨并解决上述临床问题。本课题首先通过在大鼠体内运用胆总管结扎以及50%肝脏切除手术比较正常大鼠与梗阻性黄疸大鼠肝切除术后的肝脏恢复能力。我们发现:相对于对照组,梗阻性黄疸组大鼠肝切除术后肝脏恢复能力显著下降,主要是由于肝切除术后肝再生早期肝细胞增殖能力被抑制引起。而同时梗阻性黄疸组大鼠肝再生过程中细胞因子、生长因子及肝细胞内脂质含量都发生显著紊乱。更进一步,通过对脂肪酸代谢相关基因表达的检测,我们发现它们在肝切除术后梗阻性黄疸组中表达量都出现下降(PPARα,CTP1,SREBP-1c和FASN)。因此我们认为:梗阻性黄疸引起的肝脏损伤会干扰50%肝切除术后早期肝脏再生过程中生长因子及细胞因子的表达,并减少肝切除术后早期脂肪酸的合成以及利用从而抑制肝切除术后的肝脏再生过程。这提示在临床工作中,外科医生应在肝门部胆管癌患者实行肝切除前减轻梗阻性黄疸状态引起的肝损伤,以减少肝切除术后肝功能衰竭及相关并发症的发生。而目前临床实践中应用的胆道引流方法可引起诸多并发症甚至肿瘤种植转移,因而临床工作中需要新的有效快捷的减轻梗阻性黄疸肝损伤的方法。梗阻性黄疸肝损伤的发生与肝细胞内疏水性胆汁酸的累积相关,我们发现调控胆汁酸合成底物胆固醇的关键酶:香叶基香叶基二磷酸合成酶(GGPPS),可能与梗阻性黄疸肝损伤相关。随后我们在肝门部胆管癌患者以及通过胆总管结扎构建的梗阻性黄疸小鼠中证实:伴随着梗阻性黄疸肝损伤的加重,肝脏内香叶基香叶基二磷酸合成酶(GGPPS)的表达量显著下降,提示GGPPS可能参与到梗阻性黄疸引起肝损伤的过程中。因此我们构建了小鼠肝脏特异性敲除Ggps1模型,我们发现:肝脏特异性敲除Ggps1能够有效缓解梗阻性黄疸引起的急性肝脏损伤。与对照组相比,肝脏特异性Ggps1敲除小鼠在胆总管结扎后生存率大大改善,血清AST下降,HE染色也表明敲除组小鼠肝脏形态正常,没有出现液化性坏死的;敲除小鼠肝细胞凋亡行为减少,增殖行为增加。更进一步的液相色谱质谱分析的结果表明:相对于对照组,敲除小鼠肝脏内输水性胆酸含量大大减少,提示肝脏特异性敲除Ggps1可能通过增加肝细胞内的疏水性胆酸排泄缓解梗阻性黄疸引起的肝损伤。此外,Real Time qPCR的结果也显示:相对于对照组,敲除组小鼠肝细胞内介导胆酸向肝静脉以及胆管输出的输出蛋白的表达量大大增加。而在另一方面,我们通过质谱分析发现敲除小鼠肝脏内的FOH和FPP含量相对增加,体外实验表明,FOH和FPP可以有效增加FXR的荧光素酶活性,表明FOH和FPP可以激活FXR。而针对对照组和敲除组小鼠原代肝细胞的FXR的荧光素酶活性检测也发现:敲除小鼠肝细胞内的FXR的荧光素酶活性大大增加。从而解释了肝脏特异性敲除Ggps1改善梗阻性黄疸肝损伤的具体机制。最后,鉴于肝脏特异性Ggps1敲除对改善梗阻性黄疸肝损伤的重要意义,我们对梗阻性黄疸小鼠运用了 GGPPS的一种特异性抑制剂-DGBP,我们同样发现通过皮下注射DGBP能够增加肝脏中FOH和FPP含量,并对缓解梗阻性黄疸引起的肝损伤具有一定的治疗意义。综上,我们的结果提示肝脏特异性Ggps1敲除通过增加肝细胞内的FOH和FPP含量,刺激FXR信号通路,增加转运蛋白如MRP3,BSEP,OSTβ等的表达,从而减轻梗阻性黄疸引起的肝脏损伤,为临床解决梗阻性黄疸引起的肝脏损伤提供了一个新的治疗靶点。
[Abstract]:Malignant obstructive jaundice is caused by malignant tumor growth obstruction of hepatic duct or common bile duct, bile excretion can not lead to the intestinal tract, normal enterohepatic bile cycle is blocked by the Department of hepatobiliary surgery. In practice, malignant obstructive jaundice is usually intrahepatic cholangiocarcinoma, carcinoma of head of pancreas, distal common bile duct carcinoma, especially the symptoms in patients with gallbladder carcinoma hilar bile duct carcinoma. In the surgical treatment for hilar bile duct cancer, hepatobiliary surgeons are often required for patients of hilar cholangiocarcinoma underwent liver resection to achieve radical treatment. There is a deep understanding of liver injury but failed to liver injury for obstructive jaundice would inhibit liver after resection of liver regeneration and there is no other more effective methods to improve the obstructive jaundice. So this paper intends to explore and solve the related animal experiment based on clinical problems. This paper in rats by common bile duct ligation and 50% liver resection compared to normal rat liver of rats with obstructive jaundice after resection of liver recovery. We found that: compared with the control group, the liver recovery ability decreased significantly in obstructive jaundice rats after hepatic resection, mainly due to the liver resection of liver regeneration after early liver cell proliferation was inhibited. And at the same time caused by obstructive jaundice rat liver regeneration cytokines, growth factor and lipid content in liver cells had significant disorder. Further, by detecting the expression of genes related to fatty acid metabolism, we found them in obstructive jaundice in the group of patients after liver resection was decreased (PPAR alpha, CTP1, SREBP-1c and FASN). So we think: liver injury caused by obstructive jaundice may interfere with the 50% early liver regeneration of liver resection The expression of growth factors and cytokines in the process, and reduce the liver resection in early stage after fatty acid synthesis and use to inhibit after hepatectomy in liver regeneration. In the clinical work, the surgeon should be in patients with hilar cholangiocarcinoma before implementation of liver resection to reduce liver damage caused by obstructive jaundice. In order to reduce liver failure and complications after resection. The clinical practice in the current application of biliary drainage method can cause many complications and tumor metastasis, relieve obstructive jaundice liver injury and clinical work need effective fast new. Related accumulation of bile acids and liver hydrophobic cells of obstructive jaundice liver injury, we found the key enzymes regulating bile acid synthesis substrate cholesterol: phosphate synthase (GGPPS), Kaba Kikahaji may and obstruction Jaundice associated liver injury. We then confirmed in patients with hilar cholangiocarcinoma and constructed by ligation of common bile duct obstructive jaundice in mice with obstructive jaundice liver injury, liver geranylgeranyl diphosphate synthase two (GGPPS) expression was significantly decreased, suggesting that GGPPS may be involved in obstructive jaundice cause liver damage in the process. So we constructed the mouse liver specific knockout of the Ggps1 model, we found that liver specific knockout of Ggps1 can effectively alleviate the acute liver injury caused by obstructive jaundice. Compared with the control group, liver specific Ggps1 knockout mice survival rate is improved greatly after bile duct ligation, serum AST decreased, HE staining also showed that knockout mice liver were normal, no liquefactive necrosis of liver cell apoptosis; knockout mice behavior reduced proliferation increased. Further the liquid phase GC-MS analysis showed that: compared with the control group, the water content of cholic acid knockout mice liver significantly reduced, suggesting that the liver specific knockout of Ggps1 may increase liver damage through hydrophobic bile acid excretion in liver cells of relief of obstructive jaundice caused by Real Time qPCR. In addition, the results also showed that: Compared with the control group, knockout mice liver cells in hepatic vein and to bile acid mediated protein expression and output the output of the bile duct is greatly increased. On the other hand, we found that knockdown of FOH by mass spectrometry and the content of FPP in mouse liver is increased, the in vitro experiments showed that FOH and FPP can effectively increase the luciferase activity of FXR FOH and FPP, showed that FXR. can be activated in the control group and the knock detection of luciferase activity in group mouse primary hepatocytes FXR also found that knock fluorescence in hepatocytes of FXR peptide enzyme activity Of greatly increased. Thus explaining the liver specific knockout of the specific mechanism of Ggps1 in the treatment of obstructive jaundice liver injury. Finally, in view of the liver specific Ggps1 knockout on the important meaning of improving the obstructive jaundice liver injury, we use a specific inhibitor of -DGBP GGPPS on obstructive jaundice mice, we also found that the subcutaneous injection of DGBP can increase FOH and FPP content in the liver, and has a certain significance to the treatment of liver injury caused by obstructive jaundice remission. In summary, our results suggest that liver specific Ggps1 knockout by increasing FOH and FPP content in liver cells, stimulation of the FXR signaling pathway, increase transport proteins such as MRP3, BSEP. The expression of OST beta, so as to reduce the liver injury caused by obstructive jaundice, provides a new therapeutic target for the clinical solution of obstructive jaundice caused by liver damage.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735

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