本文選題：香葉基香葉基二磷酸合成酶　切入點(diǎn)：梗阻性黃疸　出處：《南京大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：惡性梗阻性黃疸是由于惡性腫瘤生長(zhǎng)阻塞肝總管或膽總管,導(dǎo)致膽汁無(wú)法排泄至腸道,膽汁的正常肝腸循環(huán)被阻斷引起。在肝膽外科實(shí)踐中,惡性梗阻性黃疸通常是肝內(nèi)膽管癌、胰頭癌、遠(yuǎn)端膽管癌、膽囊癌尤其是肝門部膽管癌患者的伴隨癥狀。在針對(duì)肝門部膽管癌的外科治療中,肝膽外科醫(yī)生常常需要對(duì)肝門部膽管癌患者進(jìn)行肝臟大部分切除術(shù)以達(dá)到根治性治療目的。但目前對(duì)于梗阻性黃疸引起的肝損傷是否會(huì)抑制肝切除術(shù)后肝臟再生能力以及有無(wú)其他更有效的方法改善梗阻性黃疸引起的肝損傷未能有很深入的認(rèn)知。因此本課題擬基于相關(guān)動(dòng)物實(shí)驗(yàn)探討并解決上述臨床問(wèn)題。本課題首先通過(guò)在大鼠體內(nèi)運(yùn)用膽總管結(jié)扎以及50%肝臟切除手術(shù)比較正常大鼠與梗阻性黃疸大鼠肝切除術(shù)后的肝臟恢復(fù)能力。我們發(fā)現(xiàn):相對(duì)于對(duì)照組,梗阻性黃疸組大鼠肝切除術(shù)后肝臟恢復(fù)能力顯著下降,主要是由于肝切除術(shù)后肝再生早期肝細(xì)胞增殖能力被抑制引起。而同時(shí)梗阻性黃疸組大鼠肝再生過(guò)程中細(xì)胞因子、生長(zhǎng)因子及肝細(xì)胞內(nèi)脂質(zhì)含量都發(fā)生顯著紊亂。更進(jìn)一步,通過(guò)對(duì)脂肪酸代謝相關(guān)基因表達(dá)的檢測(cè),我們發(fā)現(xiàn)它們?cè)诟吻谐g(shù)后梗阻性黃疸組中表達(dá)量都出現(xiàn)下降(PPARα,CTP1,SREBP-1c和FASN)。因此我們認(rèn)為:梗阻性黃疸引起的肝臟損傷會(huì)干擾50%肝切除術(shù)后早期肝臟再生過(guò)程中生長(zhǎng)因子及細(xì)胞因子的表達(dá),并減少肝切除術(shù)后早期脂肪酸的合成以及利用從而抑制肝切除術(shù)后的肝臟再生過(guò)程。這提示在臨床工作中,外科醫(yī)生應(yīng)在肝門部膽管癌患者實(shí)行肝切除前減輕梗阻性黃疸狀態(tài)引起的肝損傷,以減少肝切除術(shù)后肝功能衰竭及相關(guān)并發(fā)癥的發(fā)生。而目前臨床實(shí)踐中應(yīng)用的膽道引流方法可引起諸多并發(fā)癥甚至腫瘤種植轉(zhuǎn)移,因而臨床工作中需要新的有效快捷的減輕梗阻性黃疸肝損傷的方法。梗阻性黃疸肝損傷的發(fā)生與肝細(xì)胞內(nèi)疏水性膽汁酸的累積相關(guān),我們發(fā)現(xiàn)調(diào)控膽汁酸合成底物膽固醇的關(guān)鍵酶:香葉基香葉基二磷酸合成酶(GGPPS),可能與梗阻性黃疸肝損傷相關(guān)。隨后我們?cè)诟伍T部膽管癌患者以及通過(guò)膽總管結(jié)扎構(gòu)建的梗阻性黃疸小鼠中證實(shí):伴隨著梗阻性黃疸肝損傷的加重,肝臟內(nèi)香葉基香葉基二磷酸合成酶(GGPPS)的表達(dá)量顯著下降,提示GGPPS可能參與到梗阻性黃疸引起肝損傷的過(guò)程中。因此我們構(gòu)建了小鼠肝臟特異性敲除Ggps1模型,我們發(fā)現(xiàn):肝臟特異性敲除Ggps1能夠有效緩解梗阻性黃疸引起的急性肝臟損傷。與對(duì)照組相比,肝臟特異性Ggps1敲除小鼠在膽總管結(jié)扎后生存率大大改善,血清AST下降,HE染色也表明敲除組小鼠肝臟形態(tài)正常,沒(méi)有出現(xiàn)液化性壞死的;敲除小鼠肝細(xì)胞凋亡行為減少,增殖行為增加。更進(jìn)一步的液相色譜質(zhì)譜分析的結(jié)果表明:相對(duì)于對(duì)照組,敲除小鼠肝臟內(nèi)輸水性膽酸含量大大減少,提示肝臟特異性敲除Ggps1可能通過(guò)增加肝細(xì)胞內(nèi)的疏水性膽酸排泄緩解梗阻性黃疸引起的肝損傷。此外,Real Time qPCR的結(jié)果也顯示:相對(duì)于對(duì)照組,敲除組小鼠肝細(xì)胞內(nèi)介導(dǎo)膽酸向肝靜脈以及膽管輸出的輸出蛋白的表達(dá)量大大增加。而在另一方面,我們通過(guò)質(zhì)譜分析發(fā)現(xiàn)敲除小鼠肝臟內(nèi)的FOH和FPP含量相對(duì)增加,體外實(shí)驗(yàn)表明,FOH和FPP可以有效增加FXR的熒光素酶活性,表明FOH和FPP可以激活FXR。而針對(duì)對(duì)照組和敲除組小鼠原代肝細(xì)胞的FXR的熒光素酶活性檢測(cè)也發(fā)現(xiàn):敲除小鼠肝細(xì)胞內(nèi)的FXR的熒光素酶活性大大增加。從而解釋了肝臟特異性敲除Ggps1改善梗阻性黃疸肝損傷的具體機(jī)制。最后,鑒于肝臟特異性Ggps1敲除對(duì)改善梗阻性黃疸肝損傷的重要意義,我們對(duì)梗阻性黃疸小鼠運(yùn)用了 GGPPS的一種特異性抑制劑-DGBP,我們同樣發(fā)現(xiàn)通過(guò)皮下注射DGBP能夠增加肝臟中FOH和FPP含量,并對(duì)緩解梗阻性黃疸引起的肝損傷具有一定的治療意義。綜上,我們的結(jié)果提示肝臟特異性Ggps1敲除通過(guò)增加肝細(xì)胞內(nèi)的FOH和FPP含量,刺激FXR信號(hào)通路,增加轉(zhuǎn)運(yùn)蛋白如MRP3,BSEP,OSTβ等的表達(dá),從而減輕梗阻性黃疸引起的肝臟損傷,為臨床解決梗阻性黃疸引起的肝臟損傷提供了一個(gè)新的治療靶點(diǎn)。
[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.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735

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