【作者】 郭永泽；

【导师】 张曼利；

【作者基本信息】 河北医科大学 ， 内科学， 2004， 硕士

【摘要】 目 的 : 肝 硬 化 终 末 期 患 者 可 并 发 肝 肺 综 合 征（hepatopulmonary syndrome,HPS），HPS 预后不良。肺内血管扩张（intrapulmonary vascular dilatation, IPVD）是其特征性病理改变，循环中或局部扩张/收缩血管物质失衡是 IPVD发生的原因之一。本课题采用胆总管结扎（common bile ductligation, CBDL）制备胆汁性肝硬化模型，检测肝硬化大鼠肺组织中内皮素-1（endothelin-1，ET-1）、前列环素（prostacyclin，PGI2）含量及 ET-1、环氧合酶-1（cyclooxygenase-1，COX-1）、环氧合酶-2（COX-2）蛋白表达水平，并同时检测血浆中ET-1、PGI2 含量及肝组织 ET-1、COX-1、COX-2 蛋白表达水平作对比，以探索它们在 IPVD 发生中的作用，并对它们之间的关系作初步分析。 方法：雄性 SD 大鼠，体重 200-250 克，随机分为 2 组：实验组、假手术（sham）组。实验组采用 CBDL 制备胆汁性肝硬化模型，实验组再设 3 个时相组：1wk CBDL 组、2wkCBDL 组、4wk CBDL 组，分别在手术后 1 周末、2 周末、4周末活杀取材。sham 组大鼠仅分离出胆总管，不作结扎和剪断，手术后 4 周末活杀取材。术后第 4 周末模型组大鼠肝组织病理切片证实有肝硬化存在。各组大鼠麻醉后右心室穿刺取血，混入抗凝剂后，分离血浆，-20℃保存；取右下肺组织，制成匀浆，离心后取上清液，-20℃保存。放免法（RIA）同 1<WP=4>中 文 摘 要时测定血浆及肺组织ET-1与PGI2的稳定代谢产物6-酮-前列腺素 F1 （6-keto-PGF1 ）的含量。取大鼠肝脏同一部分、左 α α肺下叶，用 10％中性福尔马林固定 48 小时，石蜡包埋。肝组织切片采用常规 HE 染色、Masson 三色染色，肺组织切片行常规 HE 染色，光镜下观察其病理变化。各组大鼠肝、肺组织切片行免疫组化 SP 法检测 ET-1、COX-1、COX-2 蛋白表达水平，病理图象分析系统处理切片染色结果。 结果：1.肝组织病理学表现：大鼠胆总管结扎 1 周末 ，肝细胞散在变性、坏死，汇管区小胆管增生。2 周末肝细胞灶状、片状变性、坏死，增生向肝小叶内扩展。4 周末，肝细胞多萎缩消失，肝小叶中央静脉和肝窦周围出现胶原纤维沉积，纤维间隔形成，肝小叶结构紊乱。2.肺组织病理学表现：光镜下所有大鼠肺间质无水肿，肺泡细胞无变性坏死，肺泡壁无破坏，肺泡形态正常。只有 4wk CBDL 组大鼠肺泡毛细血管扩张，肺泡间隔增宽。3.血浆和肺组织 ET-1、6-keto-PGF1 含量：sham 组、1wk CBDL 组、2wk 组、4wk α组血浆 ET-1 含量（pg/ml）分别为 119.50±11.69、179±12.67、259.36±18.91、334.08±33.34，差异有显著性（F=370.79, P＜0.001），两两比较显示，各时相组均明显高于 sham 组（P﹤0.05），各时相组之间差异有显著性（P﹤0.05）；血浆6-K-PGF1α含量（pg/ml）分别为 605.77±29.58、1122.66±85.89、1686.34±30.09、1686.34±30.09，差异有显著性（F=631.22, P＜0.001)，各时相组均明显高于 sham 组（P﹤0.05），各时相组之间差异有显著性（P﹤0.05）；肺组织 ET-1含量(ng/g)分别为 2.75±0.32、2.99±0.37、2.99±0.51、3.28±0.49，差异无显著性（F=4.66，P＞0.05）；肺组织 2<WP=5>中 文 摘 要6-keto-PGF1 含量（ng/g）分别为 38.78±1.71、42.41±1.62、 α44.31±1.29、46.41±0.53，差异有显著性 (F=96.86, P﹤0.001)，各时相组均明显高于 sham 组（P＜0.05)，各时相组之间差异有显著性（P＜0.05)。4.肺组织 ET-1、COX-1、COX-2蛋白免疫组化染色：ET-1 蛋白阳性染色位于胞浆内，在 sham组主要分布于支气管上皮细胞、肺泡动脉内皮细胞、肺泡毛细血管内皮细胞、肺泡上皮细胞，在实验组上述部位均有表达；COX-1 蛋白阳性染色位于细胞浆内，在 sham 组主要分布于支气管上皮细胞、肺门大静脉平滑肌细胞，实验组表达部位与 sham 组相同；COX-2 蛋白阳性染色位于核膜上，在sham 组主要分布于支气管周围、血管周围的巨噬细胞，实验组分布于支气管上皮细胞、肺泡上皮细胞、肺巨噬细胞、血管内皮细胞。5.肺组织 ET-1、COX-1、COX-2 蛋白表达水平：sham 组、1wk CBDL 组、2wk 组、4wk 组肺组织 ET-1 蛋白表达水平（阳性面积% 下同）分别为 10.51±5.70、11.68±7.54、8.98±4.03、13.37±8.83，差异无显著性（F=0.23, P＞0.05）；COX-1 蛋白表达水平分别为 20.48±12.81、22.65±13.36、22.93±11.19、22.41±14.41，差异无显著性（F=0.073,P＞0.05）；COX-2 蛋白表达水平分别为 6.01±3.03、22.82±14.89、40.08±14.74、65.86±15.83，差异有显著性（F=70.31,P＜0.001)。各时相组均明显高于 sham 组（P﹤0.05），各时相组之间差异有显著性（P﹤0.05）。6.肝组织 ET-1、COX-1、COX-2 蛋白免疫组化染色：ET-1 蛋白阳性染色位于胞浆内，在 sham 组主要分布在中央静脉及中央静脉周围的肝细胞胞浆内，周边逐渐减弱，在实验组上述区域也有染色更多还原

【Abstract】 Objective:Hepatopulmonary syndrome(HPS) may occur insome patients with liver cirrhosis.HPS is characterized byintrapulmonary vascular dilatation(IPVD). Using a rat model ofliver cirrhosis induced by common bile duct ligation(CBDL),weexamined plasma and lung levels of ET-1,PGI2,evaluatedexpression of ET-1, cyclooxygenase-1，cyclooxygenase-2 inlung . Methods:A total of 24 male Sprague-Dawley ratsweighing 200-250g were randomly divided into 4 grops of 6each:sham CBDL,1wkCBDL,2wkCBDL,4wkCBDL.Surgery forcommon bile duct ligation was carried in 18 rats to induce livercirrhosis under sterile conditions.The common bile bile duct wasgently exposed and doubly ligated with silk threads,and excisedcompletely between the two ties.A sham operation wasperformed in 6 rats in a similar manner to that forCBDL ,involving mobilization of the common bile duct but noligation and excision.Histological analysis conforms biliarycirrhosis by 4 wk after ligation.A blood sample of 6ml wasdrawn from the right ventricle, plasma was separated fromblood samples,and was stored at -20℃ until analysis.The right 6<WP=9>英 文 摘 要lung was quickly removed , and homogenated with glasshomogenizer. The lung homogenate was centrifugated,and wasstored at -20℃ until analysis.Plasma and lung homogenateET-1 and 6-keto-PGF1 concentrations were measured by αRIA.Livers and lungs were fixed with 10％ buffered Formalinfor 48h.Small pieces of lung and liver were paraffin embedded.Paraffin sections 5μm thick serially mounted ontoslides.Hematoxylin and eosin staining was performed on tissuesections from 18 CBDL rats to assess histological changes to thelung and liver. Expression of ET-1,COX-1,COX-2 wereexamined by immunhistochemistry. Results:1.Hepatic histological examination: Themacroscopic apprearance of the liver showed the features ofbiliary cirrhosis by 4wk after ligation.2. Pulmonary histologicalexamination:All rats have no inflammation or architecturaldistortion,except 4wk CBDL rats have the alveolus capillarydilatation.3.Plasma and lung homogenate ET-1 and6-keto-PGF1 concentrations: α Levels of plasma ET-1concentrations(pg/ml) from sham,1wk CBDL, 2wk CBDL, 4wkCBDL rats were 119.50±11.69、179±12.67、259.36±18.91、334.08±33.34 and significantly increased step by step. Levelsof plasma 6-keto-PGF1 concentrations(pg/ml) from sham,1wk αCBDL, 2wk CBDL, 4wk CBDL rats were 605.77±29.58、1122.66 ± 85.89 、 1686.34 ± 30.09 、 1686.34 ± 30.09 andsignificantly increased step by step. Lung homogenate ET-1levels（ng/g） from sham,1wk CBDL, 2wk CBDL, 4wk CBDL 7<WP=10>英 文 摘 要rats were 2.75±0.32、2.99±0.37、2.99±0.51、3.28±0.49 andthere was no significant increase from CBDL rats relative tosham values. Lung homogenate 6-keto-PGF1 levels（ng/g）from αsham,1wk CBDL, 2wk CBDL, 4wk CBDL rats were 38.78±1.71、42.41±1.62、44.31±1.29、46.41±0.53 and significantlyincreased step by step. 4. Lung ET-1 positive staining wasobserved in cytosol.In sham CBDL rats they were mainlylocated in bronchiolar epithelium,vascular endothelium,alveolarcell.In CBDL rats they were observed in the same areas. COX-1positive staining was observed in cytosol. In sham CBDL ratsthey were mainly located in the media of large pulmonary veinsand the bronchial epithelium. In CBDL rats they were observedin the same areas. COX-2 positive staining was observed innucleus. In sham CBDL rats they were mainly located inmacropages around the bronchs and vessels. In CBDL rats theywere observed in alveolar macropages and alveolarcapillaries.5.Express of ET-1 protein(positive areas%) insham,1wk CBDL, 2wk CBDL, 4wk CBDL rats were 10.51±5.70、11.68±7.54、8.98±4.03、13.37±8.83 and there was nosignifica更多还原