本文關鍵詞：部分門靜脈動脈化對大鼠動脈缺血性膽管損傷的保護作用及機制研究　出處：《南京醫(yī)科大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 門靜脈動脈化 缺血性膽管病 肝動脈 膽管損傷 動物模型

【摘要】：背景:缺血性膽管疾病是指膽管因血供受損而導致膽管損傷,統(tǒng)稱為缺血性膽管病[1]。缺血性膽管損傷的有些病因明確的,而有些病因至今仍然不甚明了。肝移植時,肝動脈的并發(fā)癥(狹窄或血栓形成)容易導致缺血性膽管損傷[2]。肝動脈化療或栓塞化療后膽管壞死和硬化性膽管炎等是常見的缺血性膽管并發(fā)癥[3]。雖然膽管的血供主要依賴于肝動脈血流,多項研究發(fā)現(xiàn),肝動脈或其分支(包括膽管周圍血管網(wǎng))和門靜脈之間也存在著大量的交通支[4,5]。在肝動脈血流出現(xiàn)中斷時,門靜脈通過這些交通支逆向灌注可能在某種程度上能夠代償肝動脈血流不足。因此,當肝動脈供血不足時,或許可以用動脈化的門靜脈通過交通支來營養(yǎng)高氧耗的膽管細胞,預防和治療缺血性膽管病。動脈化的門靜脈能否預防和治療動脈缺血性膽管損傷及其中的具體機制尚不清楚,目前臨床上僅有零星的相關臨床病例報道,但缺乏足夠的實驗研究支持。目的:1、探索構建Sprague-Dawley(SD)大鼠部分門靜脈動脈化(partial portal vein arterialization,PPVA)模型的方法。2、探索SD大鼠肝臟完全動脈缺血后的膽管損傷機制,并研究PPVA對動脈缺血性膽管損傷是否有保護作用。方法:1、通過將大鼠胃十二指腸動脈近心端與門靜脈行端側吻合構建PPVA模型。通過破壞肝臟周圍動脈側支循環(huán)、切斷大鼠肝動脈及結扎膽管周圍血管網(wǎng)以構建膽管完全動脈缺血模型(bile duct ischemia,BDI),然后再施以PPVA手術,觀察手術成功率、術后大鼠術后存活率和肝動脈門靜脈吻合口通暢情況。2、將80只SD大鼠隨機分為假手術組、PPVA組、BDI組和PPVA+BDI組等4組,術后每組分別于1天、3天和14天處死大鼠,取血清行生化檢查。取肝臟組織行常規(guī)病理檢查和透射電鏡觀察肝臟組織學及超微結構的改變。通過熒光素酶-熒光素法測定大鼠肝臟三磷酸腺苷(adenosine triphosphate,ATP)含量。通過天狼星紅染色觀察肝臟纖維化情況。熒光定量PCR法測定缺氧、膽汁轉運蛋白和炎癥相關基因的表達情況。western-blot免疫印跡法和免疫組化法檢測低氧誘導因子-1α(hypoxia inducible factor-1α,HIF-1α)、炎癥因子腫瘤壞死因子α(tumor necrosis factor-α,TNF-α)蛋白和膽汁轉運蛋白表達情況。結果:1、PPVA手術技術成功率66.7%,手術時間為25.8±1.1min,門靜脈阻斷時間為5.3±1.3min,彩超證實肝動脈門靜脈吻合口通暢率為90%。PPVA+BDI手術技術成功率70%,手術時間30.1±1.6min,門靜脈阻斷時間為4.0±1.2min,彩超證實肝動脈門靜脈吻合口通暢率為100%。2、PPVA能改善大鼠膽管動脈缺血后的肝酶損害,避免缺血后造成的膽汁性梗死、膽汁滲出及膽汁瘤形成等。PPVA能提高門靜脈血流氧含量和改善膽管動脈缺血后的肝臟ATP含量。PPVA能減輕肝臟完全動脈缺血后導致的膽管增生和纖維化反應。PCR及Western-blot結果提示,術后第3天,PPVA能明顯降低動脈缺血后肝臟HIF-1α、TNF-α和膽汁轉運蛋白的基因和蛋白表達。結論:1、建立大鼠PPVA及PPVA+BDI模型是技術可行的。熟悉大鼠肝門部解剖和經(jīng)歷一定時間的手術訓練后,能提高建立動物模型的成功率。2、PPVA手術有助于改善大鼠肝臟完全動脈缺血后的缺氧和能量障礙,對動脈缺血性膽管損傷有保護作用。
[Abstract]:Background: ischemic biliary disease refers to bile duct due to impaired blood supply due to bile duct injury, cause of injury was [1]. referred to some ischemic bile duct ischemic bile duct disease clearly, and some are still unclear. The etiology of liver transplantation, hepatic artery complications (stricture or thrombosis) easily lead to ischemic injury of bile duct [2]. hepatic artery chemotherapy or after embolism chemotherapy of bile duct necrosis and sclerosing cholangitis is a common complication of [3]. ischemic biliary duct while the blood supply mainly depends on the blood flow of hepatic artery, several studies found that the hepatic artery or its branches (including peribiliary vascular network) and portal vein are communicating branch of [4,5]. in a lot of hepatic artery blood flow interruption when the portal vein perfusion through these branches may be able to reverse the compensatory blood flow of hepatic artery insufficiency in some extent. Therefore, when the hepatic artery insufficiency, perhaps The portal vein can be used by traffic artery to nutrition high oxygen consumption of bile duct cells, prevention and treatment of ischemic biliary disease. Arterialization of portal vein can be prevented and the specific mechanism of injury and the treatment of ischemic arterial duct is unclear, at present the relevant clinical cases clinically only sporadic reports, but the lack of adequate experimental study support. Objective: To explore the construction of 1, Sprague-Dawley (SD) of partial portal vein arterialization rat (partial portal vein arterialization, PPVA) model of the method of.2, explore SD rat liver artery completely mechanism bile duct injury after ischemia, and to study whether PPVA has protective effect on ischemic injury of bile duct. Methods: 1. The gastroduodenal artery of the rat proximal end of portal vein end to side neurorrhaphy to construct the PPVA model. Through the destruction of surrounding liver artery collateral circulation, cut off the rat hepatic artery and bile duct ligation weeks In order to build a complete network of blood around the bile duct ischemia model (bile duct ischemia, BDI), and then subjected to PPVA operation, observe the success rate of surgery, postoperative survival rate of rats after hepatic artery and portal vein anastomosis patency.2, 80 SD rats were randomly divided into sham operation group, PPVA group. BDI group and PPVA+BDI group and other 4 groups after operation in each group at 1 days, 3 days and 14 days, the rats were killed, serum biochemical examination. The liver tissue for pathological examination and transmission electron microscopy observation of liver histological and ultrastructural changes of rat liver. Determination of ATP by luciferase luciferin method (adenosine triphosphate, ATP). The content of Sirius red staining observation of liver fibrosis. Determination of hypoxia related genes by fluorescence quantitative PCR, bile transport protein and inflammation of the expression of.Western-blot by Western blotting and immunohistochemistry were used to detect hypoxia induced by -1 (hypoxia inducible factor-1 alpha, alpha HIF-1 alpha), inflammatory cytokines tumor necrosis factor alpha (tumor necrosis factor- TNF- alpha, alpha) protein expression and bile transport protein. Results: 1, PPVA operation success rate was 66.7%, the operation time was 25.8 + 1.1min, portal vein blocking time was 5.3 + 1.3min. Ultrasound confirmed hepatic artery and portal vein anastomosis patency rate of 90%.PPVA+BDI surgery success rate was 70%, the operation time was 30.1 + 1.6min, portal vein occlusion time was 4 + 1.2min, ultrasound confirmed hepatic artery and portal vein anastomotic patency rate was 100%.2, PPVA can improve the liver damage of bile duct artery after ischemia in rats after ischemia, to avoid causing the biliary bile leakage and bile tumor infarction, formation of.PPVA can improve the.PPVA content of ATP in liver bile duct ischemia after portal vein blood flow and improve the oxygen content can lead to hepatic artery ischemia completely after bile duct hyperplasia and fiber The reaction of.PCR and Western-blot indicated that third days after operation, PPVA can significantly reduce the liver after ischemia HIF-1 alpha gene and protein expression of TNF- alpha and bile transport protein. Conclusion: 1. The establishment of rat PPVA model and PPVA+BDI technology is feasible. Familiar with the operation training of hilar anatomy and experience of rats after the time of the establishment of the animal model can improve the success rate of.2, PPVA operation is helpful to improve the rat liver completely after artery ischemia hypoxia and the energy barrier of ischemic bile duct injury.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R657.3

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