【作者】 李健；

【导师】 牛建昭； 陶晓华；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2005， 博士

【摘要】 目的:近年来,我国酒精性肝纤维化的发病人数呈上升的趋势。有报道表明,在持续饮酒 10-20 年的人群中,酒精性肝纤维化的发病率为 20%。如今,酒精性肝纤维化作为临床独立的肝病类型受到研究者高度重视。虽然有多家研究机构长期致力于酒精性肝纤维化的研究工作,但仍有许多病理环节、机制不为我们所知。更深入的研究工作仍需要进一步开展。为此,本实验以酒精性肝纤维化大鼠模型为实验对象,研究实验性酒精性肝纤维化的主要病理机制,探讨中医“毒损肝络”致“肝络病”的病机及物质、结构基础,为中西医结合防治酒精性肝纤维化提供理论基础和动物实验的资料。方法:1.健康 SD 大鼠共 180 只,采用“白酒-吡唑-玉米油”混合物(比例为1000:250:3)灌胃配合饲喂高脂饲料的方法制备大鼠酒精性肝纤维化动物模型。分别于造模 4w、8w、12w、14w 和 16w 随机采血,分别检测血清 ALB、TP、AST、ALT、HA、ColⅣ、LN 含量;取大鼠肝组织常规石蜡包埋、切片行 HE 染色、Mallory 染色、天狼猩红染色,常规冰冻切片行油红 O 染色、糖原染色; 2.以正常大鼠做阴性对照,采用生化分析方法检测酒精性肝纤维化早期(造模 16w),大鼠肝组织匀浆中GSH、MAD和HYP含量;采用超薄切片、透射电镜观察肝血窦结构及肝窦内皮细胞的结构特点;采用免疫组化法检测肝组织Ⅰ、Ⅳ型胶原及LN蛋白表达;采用原位杂交法检测肝组织MMP-9mRNA及TIMP-1mRNA表达;采用Western-blot法检测肝组织TGF-β1蛋白含量;分离纯化大鼠肝Kupffer Cell,采用Western-blot法检测了Kupffer Cell中TNF-α和TGF-β1蛋白的表达; 3.造模结束(16w)后,大鼠随机分成 6 组:A. 模型组(生理盐水代替药物灌胃),B. 阳性药治疗组(秋水仙碱),C. 复方鳖甲软肝片高剂量治疗组,D. 复方鳖甲软肝片中剂量治疗组,E. 复方鳖甲软肝片低剂量治疗组,F. 阴性对照组(正常大鼠生理盐水灌胃),治疗 8w后取材。采用生化分析的方法检测血清ALB、TP、AST、ALT、CⅣ、LN含量及肝组织匀浆中GSH、MAD和HYP含量;测定大鼠肝门静脉管径和肝门静脉压力;肝组织常规石蜡切片HE染色、Mallory染色;肝组织石蜡切片Ⅳ型胶原及LN免疫组化染色;肝组织TGF-β1蛋白含量Western-blot分析; 4.采用 SPSS10.0 软件对相关实验数据进行统计学处理,求平均数,作单因素方差分析。 结果:通过以上实验得到下列结果: 1.酒精性肝纤维化启动或进行阶段,血清中 AST/ALT 比值及 HA 含量显著升高; 2.酒精性肝纤维化发生过程中伴随肝细胞脂肪、糖和蛋白质代谢紊乱; 3.实验性酒精性肝纤维化与CCl4等诱发的肝纤维化存在不同的发病机制,其主要病理特征是肝窦毛细血管化和窦周纤维化; 2 复方鳖甲软肝片治疗酒精性肝纤维化的实验研究4.Kupffer Cell活化是酒精性肝纤维化启动的关键“分子事件”,TNF-α高表达是Kupffer Cell活化的分子标志;酒精性肝纤维化早期Kupffer Cell是TGF-β1的主要来源细胞;5.肝窦毛细血管化和窦周纤维化形成过程中,自由基损伤、基膜成分的过表达、基质金属降解酶及其抑制剂调节障碍等因素发挥主要作用;6.从酒精性肝病病理机制得到“毒损肝络”致肝络病的病机假说:毒损肝络致肝络病中医病机包括 3 个连续的病理阶段:络脉失养、气血郁滞、津凝痰结;7.复方鳖甲软肝片抗酒精性肝纤维化的作用机制主要为:(1)促进机体产生大量抗氧化因子,提高机体对自由基的清除能力;(2)保护肝细胞,修复肝细胞损伤,降低转氨酶;(3)显著改善肝脏微循环,从而降低肝门静脉压力,预防由于门脉高压引发的并发症;(4)显著降低动物血清中 HYP、Ⅳ型胶原及 LN 水平,因而能有效阻止肝窦毛细血管化和窦周纤维化的进程;(5)通过干预TGF-β1蛋白表达及其信号传导途径而阻断肝纤维化的进行。结论:通过统计学分析及各指标间关系的深入探讨,研究结论如下:1.酒精性肝纤维化先后经历轻度酒精性脂肪肝、中度酒精性脂肪肝、重度酒精性脂肪肝和轻度酒精性肝纤维化等病理过程,其主要特点是肝窦毛细血管化和窦周纤维化;2.酒精性肝纤维化形成过程伴随蛋白、糖、脂类物质代谢障碍以及自由基损伤、促纤维生成细胞因子过表达、胶原酶及其抑制剂比例失调、基膜成分过度生成等多种病理反应。其复杂性表明,酒精性肝纤维化存在多种病理环节,宜遵循综合疗法、多靶点作用的治疗原则;3.中医“毒损肝络”致“肝络病”的病机包含 3 个连续的病理阶段:(1)络脉失养,(2)气血郁滞,(3)津凝痰结;4.以活血化瘀和通络为立法的复方中药通过阻断多个纤维化病理环节,有效阻断了肝纤维化的进行,在抗酒精性肝纤维化方面显示出良好的治疗效果。关键词:病理机制,大鼠,肝络病,复方鳖甲软肝片, 酒精性肝纤维化更多还原

【Abstract】 Objective In China, alcoholic liver fibrosis patients have been on the rise recently. It has been reported that about 20 percent of people, who have 10 to 20 years of alcohol drinking, might exhibit alcoholic liver fibrosis. Nowadays, alcoholic liver fibrosis, as an independent disease type, is focused extensively by researchers. At present, researches on pathological mechanisms of alcoholic liver fibrosis were seldom, many mechanisms were undefined yet. So based on the alcoholic liver fibrosis rat model, we investigated the major pathological mechanisms of experimental alcoholic liver fibrosis and discuss the pathogenesis and biological basis of hepatic collateral disease (LUO BING) caused by toxin invasion hepatic collatera (GAN LUO) in order to supply the basic theory and experimental data for prevention and treatment of alcoholic liver disease by Traditional Chinese and Western Medicine combination. Methods 1. The alcoholic liver fibrosis rat model was induced by intragastric infusion of a mixture of alcoholic liquid which included alcohol, conventional corn oil and pyrazole (1000:250:3), combined with a high fatty food as previously described over a period of 16 week. We examined the concentration of ALB, TP, AST, ALT, HA and LN in serum respectively in 4, 8, 12, 14 and 16 week later. Animals were sacrificed, and livers were harvested for morphology inspection. The part samples were formalin-fixed, embedded in paraffin and stained with hematoxylin and eosin, And Mallory stain, sirius red stain were used for evaluate fibronectin and collagen expression. The other part samples were snap-frozen in melting isopentane and stained with oil red O and hepatin PAS-Shiff. 2.The level of GSH, MAD, and HYP in liver tissue was analysed by some methods as previously described in early stage of alcoholic liver fibrosis. To observe the ultrastructure of hepatic sinusoid and sinusoid endothelial cell (SEC) with the ultra-lamina slice and electron microscopy. The typeⅠ , Ⅳcollagen and LN protein in liver tissue were visualized by immunohistochemistry stain. MMP-9mRNA and TIMP-1mRNA were measured by in situ hybridization and TGF-β1 protein content in liver tissue was analyzed by western-blot. Furthermor, we freshly isolated and purified the Kupffer cell and tested the expression of TNF-α and TGF-β1 protein. 3.After challenging, the mice were divided into 6 groups, those are control group, untreated model group, Colchicine Houde treated group, high dose of FFBJRGP treated group, media dose of FFBJRGP treated group, low dose of FFBJRGP treated group. After the 8w treatment, animals were sacrificed and the contents of ALB、TP、AST、ALT、ColⅣ、LN in serum were analysed, as well as the GSH、MAD, HYP in liver tissue. The pressure and the diameter of portal vein were also examined. The part samples were formalin-fixed, embedded in paraffin and stained with hematoxylin and eosin. Mallory stain were used for evaluate collagen. The type Ⅳ collagen and LN protein in liver tissue were visualized by immunohistochemistry stain. The other part samples were homogenated to analyse the protein of TGF-β1. 4. Spss 10.0 was used. Data were analyzed using one-way ANOVA. All values were expressed as mean ± SE. Statistical significance was assigned at a P value of <0.05 or <0.01. Results 1. The proportion of AST/ALT and HA content in serum raised in the startup or progress stages of ALD. 2. The lipid, carbohydrate and protein in hepatic cell decompensate in the progress of ALD. 3. The mechanism of liver fibrosis induced by alcohol are different form CCl4, the major pathological changes of alcoholic liver fibrosis are hepatic sinusoid capillarization and perisinusoid fibrosis. 4. The activation of Kupffer Cell is the critical molecule event of the startup of alcoholic liver fibrosis and high expression of TNF-α is a better marker of activation of Kupffer Cell. In the early stage of alcoholic liver fibrosis, Kupffer Cell is the main source of TGF-β1. 5. The free radical injury, the overexpression of basement membrane and the un-graduat更多还原