HO-1在大鼠胆道缺血再灌注损伤中对胆盐转运蛋白的影响及机制研究

发布时间：2018-06-22 10:22

本文选题：血红素氧合酶-1 + 胆道　；参考：《昆明医科大学》2012年硕士论文

【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(p0.05)：siRNA组血清ALT、AST、TB、ALP含量高于对照组和空载体组(p0.05)；HO-1组明显低于siRNA组(p0.01),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(p0.05)；HO-1组血清HGF、IL-10明显高于对照组和空载体组(p0.05)；HO-1组明显高于siRNA组(p0.01),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(p0.05)；siRNA组血清IL-6、TNF-a含量高于对照组和空载体组(p0.05)；HO-1组明显低于siRNA组(p0.01),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(p0.05)；siRNA组HO-1、Ntcp、Bsep、Mrp2mRNA表达水平低于对照组和空载体组(p0.05)；HO-1组表达水平明显高于siRNA组(p0.01),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(p0.05);HO-1组Ntcp蛋白表达水平高于对照组和空载体组(p0.05)；HO-1组表达水平明显高于siRNA组(p0.01),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。
[Abstract]:Purpose :

To investigate the effect of heme oxygenase - 1 ( HO - 1 ) on the expression of heme oxygenase - 1 ( HO - 1 ) in rat liver , and to explore its effect on ischemia - reperfusion injury ( IRI ) of rat biliary epithelial cells and its possible mechanism .

Method :

1 . A 70 % liver ischemia / reperfusion injury model was established by using the method reported by X et al .

2 . Ad - HO - 1 , Ad - HO - 1 - siRNA , empty adenovirus vector and physiological saline were used to induce liver ischemia / reperfusion injury model in rats . The rats were randomly divided into 4 groups : normal saline control group ( n = 32 ) ;
Empty carrier group ( n = 32 ) ;
HO - 1 group ( n = 32 ) , the dosage of injection was 2 脳 109 TU / L , the moi = 1 ;
The levels of alanine aminotransferase ( ALT ) , aspartate aminotransferase ( AST ) , total bilirubin ( TB ) and alkaline phosphatase ( ALP ) in serum were detected after liver ischemia and reperfusion .
The levels of IL - 6IL - 10 , HGF and TNF - a in serum were determined by ELISA .
Fluorescence quantitative PCR was used to detect the expression of mRNA of bile salt output pump , the mRNA of multidrug resistance protein 2 , the expression of Ntcp mRNA and the expression of HO - 1 mRNA in liver tissue at 1d .
Western Blotting was used to detect the expression of Ntcp protein in liver tissue .
Immunofluorescent double staining and chloroacetate esterase staining were performed in the bile duct tissue of the liver and the hepatic portal , and the morphological structure of the bile duct and the surrounding liver was observed .

Results :

1 . The rat liver ischemia - reperfusion injury model was successfully performed in 128 cases , and the partial liver ischemia time was 45 min .

2 . Compared with control group and empty vector group , ALT , AST , TB and ALP were lower in HO - 1 group than those in control group and empty vector group ( P < 0.05 ) .
The HO - 1 group was significantly lower than that of siRNA group ( p0.01 ) .

3 . Compared with the control group and empty vector group , the serum HGF and IL - 10 of siRNA group were significantly lower than those in control group and empty vector group ( p < 0.05 ) .
The levels of HGF and IL - 10 in HO - 1 group were significantly higher than those in control group and empty vector group ( p < 0.05 ) .
In HO - 1 group , the levels of AIL - 6 and TNF - a were lower in HO - 1 group than in control group and empty vector group ( p < 0.05 ) .
The levels of IL - 6 and TNF - a in siRNA group were higher than those in control group and empty vector group ( p < 0.05 ) .
The HO - 1 group was significantly lower than that of siRNA group ( p0.01 ) .

4 . Compared with the control group and empty vector group , HO - 1 ( HO - 1 ) , Ntcp ( Ntcp ) , Bcl - 2 mRNA expression level in HO - 1 group were higher than those in control group and empty vector group ( p < 0.05 ) .
The mRNA expression level of HO - 1 , Ntcp , BIL - 2 mRNA in siRNA group was lower than that in control group and empty vector group ( p < 0.05 ) .
The expression level of HO - 1 group was significantly higher than that in siRNA group ( p0.01 ) .

5 . Compared with control group and empty vector group ( p < 0.05 ) , the expression level of Ntcp protein in siRNA group was lower than that of control group and empty vector group ( p < 0.05 ) .
The expression level of HO - 1 group was significantly higher than that in siRNA group ( p0.01 ) .

6 . In the same time phase point comparison , the siRNA group immunofluorescence double staining and chloroacetate esterase staining showed that the size of the bile duct epithelial cells was blurred , the basement membrane of the part was shed , the surrounding liver cells were loose , accompanied by nuclear degeneration and severe injury performance ;
The damage of HO - 1 group was light , the size of bile duct epithelial cells was clear , the continuity of basement membrane was close , and no nuclear degeneration was seen .

Conclusion :

1 . The model of rat liver heat ischemia - reperfusion injury is a stable , reliable and simple model with strong operability .

2 . Ad - HO - 1 can effectively increase the expression of HO - 1 gene in rat liver , while Ad - HO - 1 - siRNA can effectively reduce the expression of HO - 1 gene in rat liver .

3 . Ischemia / reperfusion may cause impaired liver function , and Ad - HO - 1 can effectively improve the liver function , reduce the expression of pro - inflammatory cytokines IL - 6 and TNF - a , increase the expression of the protective cytokines HGF and IL - 10 , and reduce the ischemia - reperfusion injury of large and small bile duct .

4 . HO - 1 protects the mechanism of rat biliary tract ischemia - reperfusion injury , which may be related to the induction of high expression of bile salt transporter .
The high expression of the bile salt transporter can promote the transport and excretion of bile , thereby reducing the formation of " toxic bile " with stronger cytotoxicity , and finally reducing the damage of the bile salt to the bile duct .
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363;R657.4

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