Notch信號(hào)通路在缺血再灌注損傷后膽道周?chē)軈苍偕械淖饔?/H1>
發(fā)布時(shí)間：2018-04-22 12:52
本文選題：肝缺血再灌注損傷 + 膽道周?chē)軈?/strong>�。� 參考：《昆明醫(yī)科大學(xué)》2015年碩士論文
【摘要】：[背景]缺血再灌注損傷(ischemia reperfusion injury, IRI)是指組織缺血缺氧后恢復(fù)血流與氧供,不僅未能使器官組織功能恢復(fù),反而加重組織器官功能障礙和結(jié)構(gòu)損傷；其機(jī)制復(fù)雜,它會(huì)導(dǎo)致組織直接損傷,并啟動(dòng)一連串的導(dǎo)致炎癥、細(xì)胞死亡甚至器官衰竭的有害細(xì)胞反應(yīng)。IRI對(duì)肝臟和膽道系統(tǒng)均造成不同程度的損傷,而膽道系統(tǒng)對(duì)IRI較肝實(shí)質(zhì)更敏感,因此膽道是冷、熱IRI重要的靶器官。IRI主要損傷膽道周?chē)軈?peribiliary vascular plexus, PBVP),引起PBVP微血栓形成、內(nèi)皮細(xì)胞損傷,致膽道微循環(huán)障礙；繼發(fā)的膽管炎癥、水腫、纖維化再壓迫鄰近PBVP,形成惡性循環(huán)。Notch信號(hào)通路直接接受鄰近細(xì)胞的信號(hào)并傳到細(xì)胞核,激活相關(guān)轉(zhuǎn)錄因子的表達(dá),調(diào)控多種細(xì)胞增殖、分化、凋亡,調(diào)節(jié)血管再生。多項(xiàng)通過(guò)建立大鼠心、腎等器官缺血再灌注模型的研究認(rèn)為,Notch通路具有調(diào)節(jié)缺血器官血管再生的能力,對(duì)預(yù)防IRI有重要意義。但其在缺血再灌注損傷后PBVP再生中的作用尚需進(jìn)一步研究。[目的]γ-分泌酶抑制劑DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenyl-glycine t-butyl ester)是Notch信號(hào)轉(zhuǎn)導(dǎo)途徑的特異性阻斷劑,能夠有效地抑制Notch信號(hào)通路。本研究通過(guò)建立大鼠原位自體肝移植膽道缺血再灌注損傷模型,采用DAPT對(duì)大鼠進(jìn)行預(yù)處理,分析大鼠膽道缺血再灌注后Notch信號(hào)通路相關(guān)蛋白分子在匯管區(qū)的表達(dá)情況、血清轉(zhuǎn)氨酶、組織學(xué)病理變化(匯管區(qū)血管再生情況),探討Notch信號(hào)通路在膽道缺血再灌注損傷后PBVP再生中的作用,為肝移植術(shù)后膽道并發(fā)癥的防治提供新的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。[方法]1.參照周杰等介紹的方法,建立大鼠原位自體肝移植膽道缺血再灌注損傷模型。2.SPF級(jí)健康成年雄性SD大鼠48只,體重250-280g,隨機(jī)分為3組,分別為假手術(shù)組(SO)、缺血再灌注組(IRI)、DAPT預(yù)處理組(DAPT),IRI組、DAPT組無(wú)肝期為18min,門(mén)靜脈復(fù)流后仍?shī)A閉肝動(dòng)脈30min,每組按再灌注時(shí)間不同隨機(jī)分為3天、7天兩個(gè)亞組,即SO3d、SO7d、IRI3d、IRI7d、DAPT3d、DAPT7d,每個(gè)亞組n=8；SO組：術(shù)前2h每只大鼠予以腹腔注射1ml生理鹽水,僅常規(guī)進(jìn)入腹腔后游離肝周韌帶,結(jié)扎離斷較粗的膈下靜脈、右腎上腺上靜脈,游離肝下下腔靜脈至右腎靜脈水平,游離肝十二指腸韌帶,顯露肝門(mén)結(jié)構(gòu),不予血管夾閉阻斷,關(guān)閉腹腔；IRI組為陽(yáng)性對(duì)照組,術(shù)前2h每只大鼠予以腹腔注射生理鹽水,劑量為1m1/只,然后按建模方法行膽道缺血再灌注處理;DAPT干預(yù)組：術(shù)前2h每只大鼠予以腹腔注射DAPT 4.3mg/kg,劑量為1m1/只,然后行膽道缺血再灌注處理。各組動(dòng)物經(jīng)缺血再灌注后處死,腹主動(dòng)脈采血5ml及獲取新鮮肝組織,檢測(cè)ALT、GGT、AKP、STB評(píng)價(jià)肝功能,肝組織HE染色觀察肝組織受損程度,電鏡觀察膽道上皮、血管內(nèi)皮細(xì)胞超微結(jié)構(gòu)變化,免疫組化法檢測(cè)匯管區(qū)相關(guān)蛋白表達(dá)情況。[結(jié)果]1.共成功制作大鼠自體原位肝移植膽道缺血再灌注模型32例。2.肝功能變化：與SO組相比較,IRI3d、DAPT3d、DAPT7d亞組血清ALT、ALP、 GGT、TBIL水平均明顯升高,高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05), IRI7d亞組ALP、GGT、TBIL水平明顯高于SO7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),IRI7d亞組ALT水平稍高于SO7d亞組,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.∞);與IRI組比較,DAPT3d、DAPT7d亞組血清ALT、ALP、GGT、 TBIL水平均明顯高于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。組內(nèi)亞組間比較：SO3d和S07d亞組間血清ALT、ALP、GGT、TBIL水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；IRI組和DAPT組3d亞組的血清ALT、ALP、 GGT、TBIL水平明顯高于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3匯管區(qū)CD34陽(yáng)性表達(dá)的微血管密度(MVD)的變化：與SO組相比較,IRI3d、 IRI7d、DAPT7d亞組匯管區(qū)MVD明顯高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),DAPT3d亞組與SO7d亞組的MVD差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與IRI組比較,DAPT3d、DAPT7d匯管區(qū)MVD明顯低于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；IRI組、DAPT組3d亞組的MVD水平明顯低于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4匯管區(qū)D114、Notch1、Hes1的表達(dá)情況4.1 D114的表達(dá)情況：與SO組相比,IRI3d亞組的匯管區(qū)D114蛋白水平明顯升高,高于SO3d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),IRI7d、DAPT3d、DAPT7d亞組與SO組相同時(shí)間點(diǎn)亞組D114蛋白水平的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；IRISd亞組D114蛋白表達(dá)水平明顯高于DAPT3d組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而IRI7d亞組與DAPT7d亞組D114蛋白水平差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4.2 Notchl的表達(dá)情況：與SO組相比較,IRI3d、IRI7d、DAPT3d亞組匯管區(qū)Notch1蛋白表達(dá)水平明顯高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05), DAPT7d亞組與SO7d亞組的Notch1蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與IRI組比較,DAPT3d、DAPT7d匯管區(qū)Notch1蛋白表達(dá)水平明顯低于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);SO3d和S07d亞組間Notch1蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；IRI組、DAPT組3d亞組的Notch1水平明顯高于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4.3 Hes1的表達(dá)情況：與SO組相比較,IRI3d、IRI7d、DAPT3d亞組匯管區(qū)hes1蛋白表達(dá)水平明顯高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),DAPT7d亞與SO7d亞組的hesl蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與IRI組比較,DAPT3d、DAPT7d匯管區(qū)hesl蛋白表達(dá)水平明顯低于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；SO3d和SO7d亞組間hesl蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)：IRI組、DAPT組3d亞組的Hes1水平明顯高于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.口5)。5電鏡觀察匯管區(qū)細(xì)胞超微結(jié)構(gòu)的變化：電鏡下可見(jiàn)SO組各時(shí)間點(diǎn)膽管上皮細(xì)胞和血管內(nèi)皮細(xì)胞形態(tài)結(jié)構(gòu)清晰,核膜、核仁清晰；細(xì)胞器結(jié)構(gòu)正常；IRI組及DAPT組術(shù)后第3天可見(jiàn)血管內(nèi)皮細(xì)胞損傷及膽管上皮細(xì)胞缺氧改變較SO組嚴(yán)重,且DAPT組膽管上皮及血管內(nèi)皮細(xì)胞損傷術(shù)后3天較IRI組術(shù)后第3天更重,可見(jiàn)胞核、胞質(zhì)損傷嚴(yán)重,血管腔內(nèi)可見(jiàn)異常形態(tài)紅細(xì)胞。兩組術(shù)后第7天上述損傷減輕。6HE染色觀察肝臟組織病理變化：SO組肝細(xì)胞無(wú)變性、壞死,肝小葉結(jié)構(gòu)清楚；匯管區(qū)膽管結(jié)構(gòu)清晰、完整,膽管周?chē)?xì)血管叢結(jié)構(gòu)無(wú)明顯改變。IRI組及DAPT組術(shù)后3天見(jiàn)肝小葉損傷、膽管及膽管周?chē)軈矒p傷較假手術(shù)組嚴(yán)重,而DAPT組較IRJ組損傷加重,可見(jiàn)到更多膽管淤膽及毛細(xì)血管管腔內(nèi)可見(jiàn)紅細(xì)胞淤積及微血栓形成等現(xiàn)象；且兩組術(shù)后第7天上述改變好轉(zhuǎn),而DAPT組術(shù)后7天匯管區(qū)可有有膽管增生表現(xiàn)。[結(jié)論]1、參照周杰等的方法成功建立的大鼠自體原位肝移植膽道缺血再灌注損傷模型是一個(gè)可靠的、重復(fù)性好的動(dòng)物模型,可操作性強(qiáng)。該模型有效地避免了移植術(shù)后排斥反應(yīng)、免疫抑制劑等因素對(duì)實(shí)驗(yàn)的干擾,有利于我們更加可控地對(duì)大鼠膽道缺血再灌注損傷進(jìn)行研究。2、通過(guò)檢測(cè)肝臟匯管區(qū)D114、Notch1、hes1、CD34的表達(dá)變化,結(jié)果表明：膽道缺血再灌注損傷后,Notch信號(hào)通路被激活,有效地促進(jìn)了膽道周?chē)⒀苄纬?Notch信號(hào)通路對(duì)膽道缺血再灌注損傷后膽道周?chē)軈驳脑偕哂兄匾恼{(diào)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R657.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 趙宏峰;周杰;;大鼠自體原位肝移植膽道缺血再灌注損傷模型的建立[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2006年04期
2 黃志強(qiáng);;膽道的解剖生理學(xué)與肝移植后膽道并發(fā)癥[J];中華外科雜志;2006年05期
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本文編號(hào)：1787296
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本文選題：肝缺血再灌注損傷 + 膽道周?chē)軈?/strong>�。� 參考：《昆明醫(yī)科大學(xué)》2015年碩士論文
【摘要】：[背景]缺血再灌注損傷(ischemia reperfusion injury, IRI)是指組織缺血缺氧后恢復(fù)血流與氧供,不僅未能使器官組織功能恢復(fù),反而加重組織器官功能障礙和結(jié)構(gòu)損傷；其機(jī)制復(fù)雜,它會(huì)導(dǎo)致組織直接損傷,并啟動(dòng)一連串的導(dǎo)致炎癥、細(xì)胞死亡甚至器官衰竭的有害細(xì)胞反應(yīng)。IRI對(duì)肝臟和膽道系統(tǒng)均造成不同程度的損傷,而膽道系統(tǒng)對(duì)IRI較肝實(shí)質(zhì)更敏感,因此膽道是冷、熱IRI重要的靶器官。IRI主要損傷膽道周?chē)軈?peribiliary vascular plexus, PBVP),引起PBVP微血栓形成、內(nèi)皮細(xì)胞損傷,致膽道微循環(huán)障礙；繼發(fā)的膽管炎癥、水腫、纖維化再壓迫鄰近PBVP,形成惡性循環(huán)。Notch信號(hào)通路直接接受鄰近細(xì)胞的信號(hào)并傳到細(xì)胞核,激活相關(guān)轉(zhuǎn)錄因子的表達(dá),調(diào)控多種細(xì)胞增殖、分化、凋亡,調(diào)節(jié)血管再生。多項(xiàng)通過(guò)建立大鼠心、腎等器官缺血再灌注模型的研究認(rèn)為,Notch通路具有調(diào)節(jié)缺血器官血管再生的能力,對(duì)預(yù)防IRI有重要意義。但其在缺血再灌注損傷后PBVP再生中的作用尚需進(jìn)一步研究。[目的]γ-分泌酶抑制劑DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenyl-glycine t-butyl ester)是Notch信號(hào)轉(zhuǎn)導(dǎo)途徑的特異性阻斷劑,能夠有效地抑制Notch信號(hào)通路。本研究通過(guò)建立大鼠原位自體肝移植膽道缺血再灌注損傷模型,采用DAPT對(duì)大鼠進(jìn)行預(yù)處理,分析大鼠膽道缺血再灌注后Notch信號(hào)通路相關(guān)蛋白分子在匯管區(qū)的表達(dá)情況、血清轉(zhuǎn)氨酶、組織學(xué)病理變化(匯管區(qū)血管再生情況),探討Notch信號(hào)通路在膽道缺血再灌注損傷后PBVP再生中的作用,為肝移植術(shù)后膽道并發(fā)癥的防治提供新的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。[方法]1.參照周杰等介紹的方法,建立大鼠原位自體肝移植膽道缺血再灌注損傷模型。2.SPF級(jí)健康成年雄性SD大鼠48只,體重250-280g,隨機(jī)分為3組,分別為假手術(shù)組(SO)、缺血再灌注組(IRI)、DAPT預(yù)處理組(DAPT),IRI組、DAPT組無(wú)肝期為18min,門(mén)靜脈復(fù)流后仍?shī)A閉肝動(dòng)脈30min,每組按再灌注時(shí)間不同隨機(jī)分為3天、7天兩個(gè)亞組,即SO3d、SO7d、IRI3d、IRI7d、DAPT3d、DAPT7d,每個(gè)亞組n=8；SO組：術(shù)前2h每只大鼠予以腹腔注射1ml生理鹽水,僅常規(guī)進(jìn)入腹腔后游離肝周韌帶,結(jié)扎離斷較粗的膈下靜脈、右腎上腺上靜脈,游離肝下下腔靜脈至右腎靜脈水平,游離肝十二指腸韌帶,顯露肝門(mén)結(jié)構(gòu),不予血管夾閉阻斷,關(guān)閉腹腔；IRI組為陽(yáng)性對(duì)照組,術(shù)前2h每只大鼠予以腹腔注射生理鹽水,劑量為1m1/只,然后按建模方法行膽道缺血再灌注處理;DAPT干預(yù)組：術(shù)前2h每只大鼠予以腹腔注射DAPT 4.3mg/kg,劑量為1m1/只,然后行膽道缺血再灌注處理。各組動(dòng)物經(jīng)缺血再灌注后處死,腹主動(dòng)脈采血5ml及獲取新鮮肝組織,檢測(cè)ALT、GGT、AKP、STB評(píng)價(jià)肝功能,肝組織HE染色觀察肝組織受損程度,電鏡觀察膽道上皮、血管內(nèi)皮細(xì)胞超微結(jié)構(gòu)變化,免疫組化法檢測(cè)匯管區(qū)相關(guān)蛋白表達(dá)情況。[結(jié)果]1.共成功制作大鼠自體原位肝移植膽道缺血再灌注模型32例。2.肝功能變化：與SO組相比較,IRI3d、DAPT3d、DAPT7d亞組血清ALT、ALP、 GGT、TBIL水平均明顯升高,高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05), IRI7d亞組ALP、GGT、TBIL水平明顯高于SO7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),IRI7d亞組ALT水平稍高于SO7d亞組,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.∞);與IRI組比較,DAPT3d、DAPT7d亞組血清ALT、ALP、GGT、 TBIL水平均明顯高于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。組內(nèi)亞組間比較：SO3d和S07d亞組間血清ALT、ALP、GGT、TBIL水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；IRI組和DAPT組3d亞組的血清ALT、ALP、 GGT、TBIL水平明顯高于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3匯管區(qū)CD34陽(yáng)性表達(dá)的微血管密度(MVD)的變化：與SO組相比較,IRI3d、 IRI7d、DAPT7d亞組匯管區(qū)MVD明顯高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),DAPT3d亞組與SO7d亞組的MVD差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與IRI組比較,DAPT3d、DAPT7d匯管區(qū)MVD明顯低于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；IRI組、DAPT組3d亞組的MVD水平明顯低于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4匯管區(qū)D114、Notch1、Hes1的表達(dá)情況4.1 D114的表達(dá)情況：與SO組相比,IRI3d亞組的匯管區(qū)D114蛋白水平明顯升高,高于SO3d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),IRI7d、DAPT3d、DAPT7d亞組與SO組相同時(shí)間點(diǎn)亞組D114蛋白水平的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；IRISd亞組D114蛋白表達(dá)水平明顯高于DAPT3d組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而IRI7d亞組與DAPT7d亞組D114蛋白水平差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4.2 Notchl的表達(dá)情況：與SO組相比較,IRI3d、IRI7d、DAPT3d亞組匯管區(qū)Notch1蛋白表達(dá)水平明顯高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05), DAPT7d亞組與SO7d亞組的Notch1蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與IRI組比較,DAPT3d、DAPT7d匯管區(qū)Notch1蛋白表達(dá)水平明顯低于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);SO3d和S07d亞組間Notch1蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；IRI組、DAPT組3d亞組的Notch1水平明顯高于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4.3 Hes1的表達(dá)情況：與SO組相比較,IRI3d、IRI7d、DAPT3d亞組匯管區(qū)hes1蛋白表達(dá)水平明顯高于SO組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),DAPT7d亞與SO7d亞組的hesl蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與IRI組比較,DAPT3d、DAPT7d匯管區(qū)hesl蛋白表達(dá)水平明顯低于IRI組相同時(shí)間點(diǎn)亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；SO3d和SO7d亞組間hesl蛋白表達(dá)水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)：IRI組、DAPT組3d亞組的Hes1水平明顯高于7d亞組,差異有統(tǒng)計(jì)學(xué)意義(P0.口5)。5電鏡觀察匯管區(qū)細(xì)胞超微結(jié)構(gòu)的變化：電鏡下可見(jiàn)SO組各時(shí)間點(diǎn)膽管上皮細(xì)胞和血管內(nèi)皮細(xì)胞形態(tài)結(jié)構(gòu)清晰,核膜、核仁清晰；細(xì)胞器結(jié)構(gòu)正常；IRI組及DAPT組術(shù)后第3天可見(jiàn)血管內(nèi)皮細(xì)胞損傷及膽管上皮細(xì)胞缺氧改變較SO組嚴(yán)重,且DAPT組膽管上皮及血管內(nèi)皮細(xì)胞損傷術(shù)后3天較IRI組術(shù)后第3天更重,可見(jiàn)胞核、胞質(zhì)損傷嚴(yán)重,血管腔內(nèi)可見(jiàn)異常形態(tài)紅細(xì)胞。兩組術(shù)后第7天上述損傷減輕。6HE染色觀察肝臟組織病理變化：SO組肝細(xì)胞無(wú)變性、壞死,肝小葉結(jié)構(gòu)清楚；匯管區(qū)膽管結(jié)構(gòu)清晰、完整,膽管周?chē)?xì)血管叢結(jié)構(gòu)無(wú)明顯改變。IRI組及DAPT組術(shù)后3天見(jiàn)肝小葉損傷、膽管及膽管周?chē)軈矒p傷較假手術(shù)組嚴(yán)重,而DAPT組較IRJ組損傷加重,可見(jiàn)到更多膽管淤膽及毛細(xì)血管管腔內(nèi)可見(jiàn)紅細(xì)胞淤積及微血栓形成等現(xiàn)象；且兩組術(shù)后第7天上述改變好轉(zhuǎn),而DAPT組術(shù)后7天匯管區(qū)可有有膽管增生表現(xiàn)。[結(jié)論]1、參照周杰等的方法成功建立的大鼠自體原位肝移植膽道缺血再灌注損傷模型是一個(gè)可靠的、重復(fù)性好的動(dòng)物模型,可操作性強(qiáng)。該模型有效地避免了移植術(shù)后排斥反應(yīng)、免疫抑制劑等因素對(duì)實(shí)驗(yàn)的干擾,有利于我們更加可控地對(duì)大鼠膽道缺血再灌注損傷進(jìn)行研究。2、通過(guò)檢測(cè)肝臟匯管區(qū)D114、Notch1、hes1、CD34的表達(dá)變化,結(jié)果表明：膽道缺血再灌注損傷后,Notch信號(hào)通路被激活,有效地促進(jìn)了膽道周?chē)⒀苄纬?Notch信號(hào)通路對(duì)膽道缺血再灌注損傷后膽道周?chē)軈驳脑偕哂兄匾恼{(diào)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R657.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 趙宏峰;周杰;;大鼠自體原位肝移植膽道缺血再灌注損傷模型的建立[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2006年04期
2 黃志強(qiáng);;膽道的解剖生理學(xué)與肝移植后膽道并發(fā)癥[J];中華外科雜志;2006年05期
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