【作者】 许坚吉；

【导师】 曾仲；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 硕士

【摘要】 目的：本研究通过调节肝脏血红素氧合酶-1(HO-1)的表达,来观察其对肝脏胆盐转运蛋白表达的影响,探讨其对大鼠胆道上皮细胞缺血再灌注损伤(IRI)的作用及其可能机制。方法：1.采用Fei X等报道的方法操作,建立70%肝脏缺血再灌注损伤模型。2.使用化学合成的Ad-HO-1、Ad-HO-1-siRNA,以及空腺病毒载体和生理盐水,经阴茎背静脉注射导入大鼠肝脏,24小时后开腹,建立大鼠肝脏缺血再灌注损伤模型。动物随机分为4组：生理盐水对照组(n=32)；空载体组(n=32)；HO-1组(n=32),注射剂量为2×109TU/只,MOI=1；siRNA组(n=32),注射剂量为2×109TU/只,MOI=1。各组再分别于再灌注损伤后1h、1d、1w和2w处死8只大鼠。检测肝脏缺血再灌注后血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TB)、碱性磷酸酶(ALP)水平；ELISA法检测IL-6IL-10、HGF、TNF-a在血清中的水平；荧光定量PCR检测胆盐输出泵(Bsep) mRNA、多药耐药蛋白2(Mrp2) mRNA和牛磺胆酸钠共转运蛋白(Ntcp) mRNA的表达,以及肝组织1d时相点HO-1的mRNA表达；Western Blotting检测肝组织Ntcp蛋白的表达；肝脏及肝门部胆管组织标本行免疫荧光双染及氯醋酸酯酶染色,观察大小胆管及周围肝组织形态结构的变化。结果：1.共成功实行大鼠肝脏缺血再灌注损伤模型128例,肝脏部分缺血时间为45min。2.相同时相点比较,HO-1组血清ALT、AST、TB、ALP低于对照组和空载体组(p<0.05)：siRNA组血清ALT、AST、TB、ALP含量高于对照组和空载体组(p<0.05)；HO-1组明显低于siRNA组(p<0.01),有统计学意义。3.相同时相点比较, siRNA组血清HGF、IL-10明显低于对照组和空载体组(p<0.05)；HO-1组血清HGF、IL-10明显高于对照组和空载体组(p<0.05)；HO-1组明显高于siRNA组(p<0.01),有统计学意义。HO-1组血清AIL-6、TNF-a低于对照组和空载体组(p<0.05)；siRNA组血清IL-6、TNF-a含量高于对照组和空载体组(p<0.05)；HO-1组明显低于siRNA组(p<0.01),有统计学意义。4.相同时相点比较,HO-1组HO-1、Ntcp、Bsep、Mrp2mRNA表达水平高于对照组和空载体组(p<0.05)；siRNA组HO-1、Ntcp、Bsep、Mrp2mRNA表达水平低于对照组和空载体组(p<0.05)；HO-1组表达水平明显高于siRNA组(p<0.01),有统计学意义。5.相同时相点比较,siRNA组Ntcp蛋白表达水平低于对照组和空载体组(p<0.05);HO-1组Ntcp蛋白表达水平高于对照组和空载体组(p<0.05)；HO-1组表达水平明显高于siRNA组(p<0.01),有统计学意义。6.相同时相点比较,siRNA组免疫荧光双染及氯醋酸酯酶染色上可见：大小胆管上皮细胞模糊,部分胆管基底膜脱落,周围肝细胞松散,伴核变性,严重损伤表现；HO-1组损伤表现较轻,大小胆管上皮细胞清晰,基底膜的连续性可,胆管周围肝细胞排列紧密,未见核变性。结论：1.大鼠肝脏热缺血再灌注损伤模型是一种稳定、可靠、简便的模型,可操作性强。2. Ad-HO-1能有效增加HO-1基因在大鼠肝脏的表达,而Ad-HO-1-siRNA能有效减少HO-1基因在大鼠肝脏的表达。3.缺血再灌注会造成肝功能受损,Ad-HO-1可有效改善肝功能,减少促炎性细胞因子IL-6、TNF-a的表达,增加了保护性细胞因子HGF、IL-10的表达,减轻了大、小胆管的缺血再灌注损伤。4.HO-1保护大鼠胆道缺血再灌注损伤的机制,可能与诱导胆盐转运蛋白高表达有关；胆盐转运蛋白高表达可以促进胆汁的转运和排泄,从而减少具有较强细胞毒性的“毒性胆汁”形成,最终减少胆盐对胆管的损伤。更多还原

【Abstract】 ObjectiveIn this study, by regulating the expression of liver heme oxygenase-1(HO-1), to observe the expression of hepatic bile salt transport protein by HO-1, Explore the rat biliary epithelial cell in ischemia and reperfusion injury (IRI) and its possible mechanismsMethods1. To establish the models of70%part of the liver ischemia-reperfusion injury by the method of operation which Fei X had reported.2. The use of chemical synthesis of Ad-HO-1, Ad-HO-1-of siRNA, empty adenoviruses and saline, into the donor liver by Genital dorsal vein. laparotomy after24hours, establish rat liver ischemia-reperfusion injury model. The animals were randomly divided into four groups:NaCl group, Empty adenovirus group, Amplification of HO-1adenovirus group, Silencing HO-1adenovirus group. To Collect specimens on1hour,1day,1week and2weeks after operation. To detect the levels of ALT、AST、TB、ALP in serum after ischemia reperfusion injury of liver. The function of liver was also evaluated. The expressions of IL-6、IL-10、HGF and TNF-a in the liver were detected. The mRNA and protein expressions of salt transporter proteins(Sodium taurocholate cotransporting polypeptide, Bile salt expot pump、Multidrug resistance-associated protein-2、sodium taurocholate cotransporting polypeptide and the HO-1mRNA expressions of liver tissue of levels in1d were measured. The livere and biliary bile duct were detected by immunofluorescence double staining and Chloro-acetate esterase staining. Observe the morphological changes in the structure of the size of the bile duct and the surrounding liver tissue.Results1.128cases of ischemia-reperfusion injury models of liver were Successfully established and partial liver ischemia time was45minutes.2. Compared with the same time points, serum ALT, AST, TB, and the ALP in HO-1groups was significantly lower than in NaCl and Adenovirus groups (p<0.05). serum ALT, AST, TB, and the ALP in siRNA groups was significantly higher than in NaCl and Adenovirus groups (p<0.05). HO-1groups was significantly lower than the siRNA groups (p<0.01).3. Compared with the same time points, siRNA groups serum HGF and IL-10was significantly lower than in NaCl and Adenovirus groups (p<0.05); HO-1groups serum HGF and IL-10was significantly higher than in NaCl and Adenovirus groups (p<0.05). HO-1groups was significantly higher than the siRNA groups (p<0.01). serum IL-6and TNF-a in G3was significantly lower than in NaCl and Adenovirus groups (p<0.05). serum IL-6and TNF-a in siRNA groups was significantly higher than in NaCl and Adenovirus groups (p<0.05). HO-1groups was significantly lower than the siRNA groups (p<0.01).4. Compared with the same time points, HO-1groups of HO-1, Ntcp, Bsep, Mrp2mRNA expression level was significantly higher than in NaCl and Adenovirus groups (p<0.05); siRNA groups of HO-1, Ntcp, Bsep, Mrp2mRNA expression level was significantly lower than in NaCl and Adenovirus groups (p<0.05); HO-1groups was significantly higher than the siRNA groups (p<0.01).5. siRNA groups Ntcp protein expression levels were significantly lower than the NaCl and Adenovirus groups (p<0.05). HO-1groups Ntcp protein expression levels were significantly higher than the NaCl and Adenovirus groups (p<0.05). HO-1groups was significantly higher than the siRNA groups (p<0.01).6. Compared with the same time points, Immunofluorescence double staining and chloro-acetate esterase staining in siRNA groups can be seen:The size of the bile duct epithelial cells is fuzzy, part of the bile duct basement membrane shedding, liver cells around loose, with degeneration, serious injury performance. The HO-1groups was less severe injury, size of the bile duct epithelial cells is clear, the continuity of the basement membrane is better, around the bile duct liver cells closely arranged, no degenerationConclusions:1. Rat liver warm ischemia reperfusion injury model is a stable, reliable, simple, the model is workable.2. Ad-HO-1can effectively increase HO-1gene expression in rat liver; Ad-HO-1-siRNA can effectively reduce the expression of HO-1gene in rat liver.3. The liver function damaged by Ischemia and reperfusion. Ad-HO-1can effectively improve liver function. Reduce the proinflammatory cytokine IL-6, TNF-a expression, an increase of protective cytokines of HGF, IL-10expression. Reduce the size of the bile duct ischemia and reperfusion injury.4. The mechanism of HO-1protect biliary ischemia-reperfusion injury in rats, may be induced by bile salt transporter protein high expression. Bile salt transporter protein expression can contribute to the transport and excretion of bile, reduce the strong cytotoxic bile formation, ultimately reduce the role of bile salts of the bile duct.更多还原