部分门静脉动脉化对大鼠动脉缺血性胆管损伤的保护作用及机制研究

发布时间：2018-01-12 05:14

本文关键词：部分门静脉动脉化对大鼠动脉缺血性胆管损伤的保护作用及机制研究　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:缺血性胆管疾病是指胆管因血供受损而导致胆管损伤,统称为缺血性胆管病[1]。缺血性胆管损伤的有些病因明确的,而有些病因至今仍然不甚明了。肝移植时,肝动脉的并发症(狭窄或血栓形成)容易导致缺血性胆管损伤[2]。肝动脉化疗或栓塞化疗后胆管坏死和硬化性胆管炎等是常见的缺血性胆管并发症[3]。虽然胆管的血供主要依赖于肝动脉血流,多项研究发现,肝动脉或其分支(包括胆管周围血管网)和门静脉之间也存在着大量的交通支[4,5]。在肝动脉血流出现中断时,门静脉通过这些交通支逆向灌注可能在某种程度上能够代偿肝动脉血流不足。因此,当肝动脉供血不足时,或许可以用动脉化的门静脉通过交通支来营养高氧耗的胆管细胞,预防和治疗缺血性胆管病。动脉化的门静脉能否预防和治疗动脉缺血性胆管损伤及其中的具体机制尚不清楚,目前临床上仅有零星的相关临床病例报道,但缺乏足够的实验研究支持。目的:1、探索构建Sprague-Dawley(SD)大鼠部分门静脉动脉化(partial portal vein arterialization,PPVA)模型的方法。2、探索SD大鼠肝脏完全动脉缺血后的胆管损伤机制,并研究PPVA对动脉缺血性胆管损伤是否有保护作用。方法:1、通过将大鼠胃十二指肠动脉近心端与门静脉行端侧吻合构建PPVA模型。通过破坏肝脏周围动脉侧支循环、切断大鼠肝动脉及结扎胆管周围血管网以构建胆管完全动脉缺血模型(bile duct ischemia,BDI),然后再施以PPVA手术,观察手术成功率、术后大鼠术后存活率和肝动脉门静脉吻合口通畅情况。2、将80只SD大鼠随机分为假手术组、PPVA组、BDI组和PPVA+BDI组等4组,术后每组分别于1天、3天和14天处死大鼠,取血清行生化检查。取肝脏组织行常规病理检查和透射电镜观察肝脏组织学及超微结构的改变。通过荧光素酶-荧光素法测定大鼠肝脏三磷酸腺苷(adenosine triphosphate,ATP)含量。通过天狼星红染色观察肝脏纤维化情况。荧光定量PCR法测定缺氧、胆汁转运蛋白和炎症相关基因的表达情况。western-blot免疫印迹法和免疫组化法检测低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、炎症因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)蛋白和胆汁转运蛋白表达情况。结果:1、PPVA手术技术成功率66.7%,手术时间为25.8±1.1min,门静脉阻断时间为5.3±1.3min,彩超证实肝动脉门静脉吻合口通畅率为90%。PPVA+BDI手术技术成功率70%,手术时间30.1±1.6min,门静脉阻断时间为4.0±1.2min,彩超证实肝动脉门静脉吻合口通畅率为100%。2、PPVA能改善大鼠胆管动脉缺血后的肝酶损害,避免缺血后造成的胆汁性梗死、胆汁渗出及胆汁瘤形成等。PPVA能提高门静脉血流氧含量和改善胆管动脉缺血后的肝脏ATP含量。PPVA能减轻肝脏完全动脉缺血后导致的胆管增生和纤维化反应。PCR及Western-blot结果提示,术后第3天,PPVA能明显降低动脉缺血后肝脏HIF-1α、TNF-α和胆汁转运蛋白的基因和蛋白表达。结论:1、建立大鼠PPVA及PPVA+BDI模型是技术可行的。熟悉大鼠肝门部解剖和经历一定时间的手术训练后,能提高建立动物模型的成功率。2、PPVA手术有助于改善大鼠肝脏完全动脉缺血后的缺氧和能量障碍,对动脉缺血性胆管损伤有保护作用。
[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.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R657.3

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