Notch信号通路在缺血再灌注损伤后胆道周围血管丛再生中的作用

发布时间：2018-04-22 12:52

本文选题：肝缺血再灌注损伤 + 胆道周围血管丛　；参考：《昆明医科大学》2015年硕士论文

【摘要】：[背景]缺血再灌注损伤(ischemia reperfusion injury, IRI)是指组织缺血缺氧后恢复血流与氧供,不仅未能使器官组织功能恢复,反而加重组织器官功能障碍和结构损伤；其机制复杂,它会导致组织直接损伤,并启动一连串的导致炎症、细胞死亡甚至器官衰竭的有害细胞反应。IRI对肝脏和胆道系统均造成不同程度的损伤,而胆道系统对IRI较肝实质更敏感,因此胆道是冷、热IRI重要的靶器官。IRI主要损伤胆道周围血管丛(peribiliary vascular plexus, PBVP),引起PBVP微血栓形成、内皮细胞损伤,致胆道微循环障碍；继发的胆管炎症、水肿、纤维化再压迫邻近PBVP,形成恶性循环。Notch信号通路直接接受邻近细胞的信号并传到细胞核,激活相关转录因子的表达,调控多种细胞增殖、分化、凋亡,调节血管再生。多项通过建立大鼠心、肾等器官缺血再灌注模型的研究认为,Notch通路具有调节缺血器官血管再生的能力,对预防IRI有重要意义。但其在缺血再灌注损伤后PBVP再生中的作用尚需进一步研究。[目的]γ-分泌酶抑制剂DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenyl-glycine t-butyl ester)是Notch信号转导途径的特异性阻断剂,能够有效地抑制Notch信号通路。本研究通过建立大鼠原位自体肝移植胆道缺血再灌注损伤模型,采用DAPT对大鼠进行预处理,分析大鼠胆道缺血再灌注后Notch信号通路相关蛋白分子在汇管区的表达情况、血清转氨酶、组织学病理变化(汇管区血管再生情况),探讨Notch信号通路在胆道缺血再灌注损伤后PBVP再生中的作用,为肝移植术后胆道并发症的防治提供新的理论基础和实验依据。[方法]1.参照周杰等介绍的方法,建立大鼠原位自体肝移植胆道缺血再灌注损伤模型。2.SPF级健康成年雄性SD大鼠48只,体重250-280g,随机分为3组,分别为假手术组(SO)、缺血再灌注组(IRI)、DAPT预处理组(DAPT),IRI组、DAPT组无肝期为18min,门静脉复流后仍夹闭肝动脉30min,每组按再灌注时间不同随机分为3天、7天两个亚组,即SO3d、SO7d、IRI3d、IRI7d、DAPT3d、DAPT7d,每个亚组n=8；SO组：术前2h每只大鼠予以腹腔注射1ml生理盐水,仅常规进入腹腔后游离肝周韧带,结扎离断较粗的膈下静脉、右肾上腺上静脉,游离肝下下腔静脉至右肾静脉水平,游离肝十二指肠韧带,显露肝门结构,不予血管夹闭阻断,关闭腹腔；IRI组为阳性对照组,术前2h每只大鼠予以腹腔注射生理盐水,剂量为1m1/只,然后按建模方法行胆道缺血再灌注处理;DAPT干预组：术前2h每只大鼠予以腹腔注射DAPT 4.3mg/kg,剂量为1m1/只,然后行胆道缺血再灌注处理。各组动物经缺血再灌注后处死,腹主动脉采血5ml及获取新鲜肝组织,检测ALT、GGT、AKP、STB评价肝功能,肝组织HE染色观察肝组织受损程度,电镜观察胆道上皮、血管内皮细胞超微结构变化,免疫组化法检测汇管区相关蛋白表达情况。[结果]1.共成功制作大鼠自体原位肝移植胆道缺血再灌注模型32例。2.肝功能变化：与SO组相比较,IRI3d、DAPT3d、DAPT7d亚组血清ALT、ALP、 GGT、TBIL水平均明显升高,高于SO组相同时间点亚组,差异有统计学意义(P0.05), IRI7d亚组ALP、GGT、TBIL水平明显高于SO7d亚组,差异有统计学意义(P0.05),IRI7d亚组ALT水平稍高于SO7d亚组,差异无统计学意义(P0.∞);与IRI组比较,DAPT3d、DAPT7d亚组血清ALT、ALP、GGT、 TBIL水平均明显高于IRI组相同时间点亚组,差异有统计学意义(P0.05)。组内亚组间比较：SO3d和S07d亚组间血清ALT、ALP、GGT、TBIL水平比较差异无统计学意义(P0.05)；IRI组和DAPT组3d亚组的血清ALT、ALP、 GGT、TBIL水平明显高于7d亚组,差异有统计学意义(P0.05)。3汇管区CD34阳性表达的微血管密度(MVD)的变化：与SO组相比较,IRI3d、 IRI7d、DAPT7d亚组汇管区MVD明显高于SO组相同时间点亚组,差异有统计学意义(P0.05),DAPT3d亚组与SO7d亚组的MVD差异无统计学意义(P0.05)；与IRI组比较,DAPT3d、DAPT7d汇管区MVD明显低于IRI组相同时间点亚组,差异有统计学意义(P0.05)；IRI组、DAPT组3d亚组的MVD水平明显低于7d亚组,差异有统计学意义(P0.05)。4汇管区D114、Notch1、Hes1的表达情况4.1 D114的表达情况：与SO组相比,IRI3d亚组的汇管区D114蛋白水平明显升高,高于SO3d亚组,差异有统计学意义(P0.05),IRI7d、DAPT3d、DAPT7d亚组与SO组相同时间点亚组D114蛋白水平的差异无统计学意义(P0.05)；IRISd亚组D114蛋白表达水平明显高于DAPT3d组,差异有统计学意义(P0.05),而IRI7d亚组与DAPT7d亚组D114蛋白水平差异无统计学意义(P0.05)。4.2 Notchl的表达情况：与SO组相比较,IRI3d、IRI7d、DAPT3d亚组汇管区Notch1蛋白表达水平明显高于SO组相同时间点亚组,差异有统计学意义(P0.05), DAPT7d亚组与SO7d亚组的Notch1蛋白表达水平比较差异无统计学意义(P0.05)；与IRI组比较,DAPT3d、DAPT7d汇管区Notch1蛋白表达水平明显低于IRI组相同时间点亚组,差异有统计学意义(P0.05);SO3d和S07d亚组间Notch1蛋白表达水平比较差异无统计学意义(P0.05)；IRI组、DAPT组3d亚组的Notch1水平明显高于7d亚组,差异有统计学意义(P0.05)。4.3 Hes1的表达情况：与SO组相比较,IRI3d、IRI7d、DAPT3d亚组汇管区hes1蛋白表达水平明显高于SO组相同时间点亚组,差异有统计学意义(P0.05),DAPT7d亚与SO7d亚组的hesl蛋白表达水平比较差异无统计学意义(P0.05)；与IRI组比较,DAPT3d、DAPT7d汇管区hesl蛋白表达水平明显低于IRI组相同时间点亚组,差异有统计学意义(P0.05)；SO3d和SO7d亚组间hesl蛋白表达水平比较差异无统计学意义(p0.05)：IRI组、DAPT组3d亚组的Hes1水平明显高于7d亚组,差异有统计学意义(P0.口5)。5电镜观察汇管区细胞超微结构的变化：电镜下可见SO组各时间点胆管上皮细胞和血管内皮细胞形态结构清晰,核膜、核仁清晰；细胞器结构正常；IRI组及DAPT组术后第3天可见血管内皮细胞损伤及胆管上皮细胞缺氧改变较SO组严重,且DAPT组胆管上皮及血管内皮细胞损伤术后3天较IRI组术后第3天更重,可见胞核、胞质损伤严重,血管腔内可见异常形态红细胞。两组术后第7天上述损伤减轻。6HE染色观察肝脏组织病理变化：SO组肝细胞无变性、坏死,肝小叶结构清楚；汇管区胆管结构清晰、完整,胆管周围毛细血管丛结构无明显改变。IRI组及DAPT组术后3天见肝小叶损伤、胆管及胆管周围血管丛损伤较假手术组严重,而DAPT组较IRJ组损伤加重,可见到更多胆管淤胆及毛细血管管腔内可见红细胞淤积及微血栓形成等现象；且两组术后第7天上述改变好转,而DAPT组术后7天汇管区可有有胆管增生表现。[结论]1、参照周杰等的方法成功建立的大鼠自体原位肝移植胆道缺血再灌注损伤模型是一个可靠的、重复性好的动物模型,可操作性强。该模型有效地避免了移植术后排斥反应、免疫抑制剂等因素对实验的干扰,有利于我们更加可控地对大鼠胆道缺血再灌注损伤进行研究。2、通过检测肝脏汇管区D114、Notch1、hes1、CD34的表达变化,结果表明：胆道缺血再灌注损伤后,Notch信号通路被激活,有效地促进了胆道周围微血管形成,Notch信号通路对胆道缺血再灌注损伤后胆道周围血管丛的再生具有重要的调控作用。
[Abstract]:[background] ischemia reperfusion injury (ischemia reperfusion injury, IRI) refers to the recovery of blood flow and oxygen supply after ischemia and hypoxia. It not only fails to restore organ function, but aggravates tissue organ dysfunction and structural damage, and its mechanism is complex, which can lead to tissue direct injury and initiate a series of inflammation and cell death. Even the harmful cell reaction of organ failure.IRI causes different degrees of damage to the liver and the biliary tract system, and the biliary tract is more sensitive to IRI than the liver parenchyma, so the biliary tract is cold. The important target organ of the hot IRI is that.IRI mainly damages the peripheral vascular plexus of the biliary tract (peribiliary vascular plexus, PBVP), resulting in the formation of PBVP microthrombus and endothelial cell damage. Injury, induced biliary microcirculation disorder, secondary bile duct inflammation, edema, and fibrosis and recompression of adjacent PBVP, forming a vicious cycle.Notch signaling pathway to receive direct signal from adjacent cells and transmitting to the nucleus, activating the expression of related transcription factors and regulating the proliferation, differentiation, apoptosis and regulation of vascular regeneration. The study of renal ischemia reperfusion model suggests that Notch pathway has the ability to regulate vascular regeneration in ischemic organs and is of great significance for the prevention of IRI. However, the role of PBVP in the regeneration of PBVP after ischemia-reperfusion injury still needs further study. [Objective] gamma secretase inhibitor DAPT (3,5-Difluorophenacetyl-L-alanyl)]-S-phenyl-glyc Ine t-butyl ester is a specific blocking agent for Notch signal transduction pathway, which can effectively inhibit the Notch signaling pathway. In this study, a rat model of biliary ischemia reperfusion injury in situ autograft in rats was established, and DAPT was used to pretreat rats. The protein molecules related to Notch signaling pathway in the rat bile duct ischemia-reperfusion were analyzed. The expression of the tube area, the serum transaminase, the histological pathological changes (the vascular regeneration in the pipe area), to explore the role of Notch signaling pathway in the regeneration of PBVP after the biliary tract ischemia reperfusion injury, and provide a new theoretical basis and experimental basis for the prevention and treatment of biliary complications after liver transplantation. [method]1. referred to the method introduced by Zhou Jie and so on. Rat model.2.SPF healthy adult male SD rats, 48 healthy adult male SD rats, were randomly divided into 3 groups: the sham operation group (SO), the ischemia reperfusion group (IRI), the DAPT preconditioning group (DAPT), the IRI group, the DAPT group without liver stage 18min, and the hepatic artery 30min after the portal vein reflow, each group was reperfused by reperfusion. The time was divided randomly into 3 days and two subgroups of 7 days, namely, SO3d, SO7d, IRI3d, IRI7d, DAPT3d, DAPT7d, each subgroup n=8; SO group: 2h rats were injected intraperitoneally with 1ml saline, only the normal intraperitoneal intraperitoneal ligaments were free, the lower inferior phrenic vein, the right superior inferior vena cava and the right kidney were ligated and detached from the inferior phrenic vein. The venous level, the hepato duodenal ligament, the hepatic portal structure, the occlusion of the blood vessel and the abdominal cavity closed, the IRI group was the positive control group, and the rats were injected with the saline in the abdominal cavity and the dose of 1m1/ only before 2h, and then the model of the bile duct ischemia and reperfusion was done according to the modeling method; the DAPT intervention group was given intraperitoneal injection of 2H before the operation. DAPT 4.3mg/kg, dose 1m1/ only, and then bile duct ischemia reperfusion treatment. All animals were killed after ischemia-reperfusion. After ischemia and reperfusion, 5ml and fresh liver tissue were collected from the abdominal aorta. ALT, GGT, AKP, STB were used to evaluate the liver function. The degree of liver tissue damage was observed by HE staining in liver tissue. The ultrastructural changes of bile duct epithelium and vascular endothelial cells were observed by electron microscopy. Immunohistochemical method was used to detect the expression of related protein in the manifold area. [results]1. 32 cases of.2. liver function change were successfully made in the rat model of biliary ischemia reperfusion in the rat orthotopic liver transplantation. Compared with the SO group, the levels of ALT, ALP, GGT and TBIL in the serum of IRI3d, DAPT3d and DAPT7d subgroups were significantly higher than those in the same time point group in the SO group, and the difference was statistically significant. Meaning (P0.05), the level of ALP, GGT and TBIL in IRI7d subgroup was significantly higher than that of SO7d subgroup, the difference was statistically significant (P0.05), the ALT level of IRI7d subgroup was slightly higher than that of the SO7d subgroup, and the difference was not statistically significant (P0. infinity). Significance (P0.05). Comparison between groups in the group: there was no significant difference in serum ALT, ALP, GGT and TBIL levels between the SO3d and S07d subgroups, and the level of serum ALT in the IRI group and DAPT group 3D subgroup was significantly higher than that of the subgroup, and the difference was statistically significant: Compared with group SO, the MVD of IRI3d, IRI7d, DAPT7d subgroup was significantly higher than that of group SO in the same time point subgroup, the difference was statistically significant (P0.05), DAPT3d subgroup and SO7d subgroup had no statistical significance (P0.05), and compared with the IRI group, the difference was significantly lower than that of the same time point group. IRI group, DAPT group 3D subgroup MVD level was significantly lower than the 7d subgroup, the difference was statistically significant (P0.05).4 sinks D114, Notch1, Hes1 expression of the 4.1 D114 expression: compared with the SO group, the level of protein level in the subgroup was significantly higher than that in the subgroup. There was no significant difference in the level of D114 protein in the subgroup of the same time point group with the SO group (P0.05), and the expression level of D114 protein in the IRISd subgroup was significantly higher than that in the DAPT3d group, and the difference was statistically significant (P0.05), but there was no significant difference between the IRI7d subgroup and the DAPT7d subgroup in the D114 protein level (P0.05). The expression level of Notch1 protein in RI7d, DAPT3d subgroup was significantly higher than that of group SO in the same time point subgroup, and the difference was statistically significant (P0.05). There was no significant difference in the level of Notch1 protein expression in DAPT7d subgroup and SO7d subgroup (P0.05), and the expression level of the protein in DAPT7d manifold was significantly lower than that in IRI group. There was significant difference in the same time point subgroup (P0.05), and there was no significant difference in the expression of Notch1 protein between SO3d and S07d subgroups (P0.05), and in group IRI, the Notch1 level of 3D subgroup of DAPT group was significantly higher than that of the 7d subgroup, and the difference was statistically significant (P0.05). The expression level of Hes1 protein was significantly higher than that in the same time point subgroup of SO group, the difference was statistically significant (P0.05). There was no significant difference in hesl protein expression level between DAPT7d subgroup and SO7d subgroup (P0.05). Compared with IRI group, the expression level of hesl protein in DAPT3d and DAPT7d sinks was significantly lower than that of the same time point group in the IRI group, and the difference was statistically significant. There was no significant difference in the level of hesl protein expression between the SO3d and SO7d subgroups (P0.05):IRI group, and the Hes1 level of 3D subgroup of DAPT group was significantly higher than that of the 7d subgroup, and the difference was statistically significant (P0. mouth 5). The ultrastructure changes of the sinks were observed in the.5 electron microscope: the bile duct epithelial cells and blood were visible at each time point under electron microscope. The endothelial cell morphology and structure were clear, the nuclear membrane and nucleolus were clear and the organelle structure was normal. The vascular endothelial cell damage and the anoxic changes of the bile duct epithelial cells in group IRI and group DAPT were more serious than that in group SO third days after operation, and the 3 days after the operation of the bile duct and vascular endothelial cells in group DAPT were heavier than those in the IRI group, and the nucleus and cytoplasmic loss were found. The injury was serious and abnormal morphologic red cells were found in the endovascular. The pathological changes of the liver were observed by.6HE staining on the seventh day after operation in two groups: no degeneration, necrosis, clear structure of hepatic lobule in group SO, clear and complete structure of bile duct in the pipe area, and no obvious changes of the structure of capillary plexus around the bile duct in group.IRI and group DAPT 3 days after operation The injury of hepatic lobule, the injury of bile duct and bile duct surrounding the vascular plexus was more serious than that of the sham operation group, but the injury of group DAPT was more serious than that of the IRJ group. More bile duct cholestasis and the formation of red blood cell deposition and microthrombus were seen in the bile duct and capillary tube, and the changes of the above two groups were improved on the seventh day after operation, and the bile duct hyperplasia in the 7 days after the operation of group DAPT could have bile duct hyperplasia. [conclusion]1, the model of rat orthotopic liver ischemia reperfusion injury established by Zhou Jie and other methods is a reliable and reproducible animal model with strong maneuverability. This model effectively avoids the rejection after transplantation and the interference of immunosuppressive agents on the experiment, which is beneficial to us. .2, the expression changes of D114, Notch1, Hes1, CD34 in the hepatic duct area were detected by control. The results showed that the Notch signaling pathway was activated after the ischemia reperfusion injury of the bile duct, which effectively promoted the formation of microvessels around the biliary tract, and the Notch signal pathway was the biliary week after the bile duct ischemia reperfusion injury. The regeneration of the peri vascular plexus has an important regulatory effect.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3

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