【作者】 王桂杰；

【导师】 李岩；

【作者基本信息】 中国医科大学 ， 内科学， 2006， 硕士

【摘要】 目的 我国是乙型肝炎发病率较高的国家,肝炎不经有效治疗进展为肝纤维化是它的发展趋势之一,而肝纤维化经适当治疗后可逆转,本文通过测定Hyp、HA、TGFβ₁和Smad2/3在四氯化碳（CCL₄）致肝纤维化的Wistar大鼠肝组织中的表达,探讨中药方剂软肝宁的疗效。 材料和方法 选取体重160-260gWistar大鼠50只,雌雄各半,随机分为正常组、模型组、治疗组和预防组4组,除正常组外其余三组均用40%CCL₄造模8周（0.2ml/100g）。预防组造模1周后中药灌胃,治疗组造模4周后中药灌胃（0.3ml/100g）。8周末处死大鼠留取肝脾组织,每组选取5个以上标本分别经HE染色后作病理分级,测定肝重及脾重并计算肝指数、脾指数以确定不同组别大鼠应用中药后的变化,应用碱水解法测定肝组织羟脯胺酸（Hyp）;应用酶联免疫法（ELISA）测定肝组织透明质酸（HA）。应用链菌素亲生物素-过氧化物酶（S-P）免疫组化染色和免疫印迹法（WesternBlot）测定肝组织中TGFβ₁和Smad2/3蛋白的表达,免疫组化抗体浓度为1:200,免疫印迹一抗浓度为1:400,二抗浓度为1:500,免疫组化染色和免疫印迹结果经计算机显微镜图像分析系统分析量化,所有实验数据用（?）±s表示,进行t检验。 结果 一般状态:8周末正常组同实验前,CCL₄模型组实验结束时毛色变黄,更多还原

【Abstract】 ObjectiveThe attack rate of type B hepatitis was high in China. One of the trend of hepatitis is hepatic fibrosis if it isnt treated effectively. Hepatic fibrosis was reversible through appropriate treatment. This paper discussed the effect of the traditional Chinese medicine called RuanGangning on the expression of Hyp, HA, TGFβ₁, and Smad2/3 in Wistar rat liver tissue. The aninal model was established by infusion of CCL4.Materials and MethodsFifty rats were divided into 4 study groups randomly, ie. normal group, model group, treatment group and prevention group. The weight ranged from 160 -260g, the number of female and male was the same. The rats model were built by infusion of 40% CCL4（0. 2ml/100g）for eight weeks except the normal group. The prevention group were lavaged with Chinese drug after one week. The treatment group were lavaged with Chinese drug after 4 weeks （0. 3ml/100g）. The rats were killed after 8 weeks to collect the liver tissue. More than 5 specimens in each group were selected for pathological grade respectively after HE stain, and the weight of liver and spleen were measured. We evalulated the index of liver and spleen in order to determine the change of each group.Hyp was detected by the method of alkaline hydrolysis and HA was detected by the method of ELISA. The expression of TGFβ₁ and Smad2/3 in hepatic tissue was detected by the method of S - P immunohistochemistry stain and Western -Blot. The concentration of antibody for immunohistochemistry was 1’ 200. The concentration of antibody No. 1 for immunoblot was 1:400 and the concentration of antibody No. 2 was 1: 500. The results were analyzed and quantitied by the program of computer microscopic image analysis. All the data was expressed by x ± s and analyzed by t test.ResultsGeneral status: Normal group was unchanged. The color pattern in rats of CCL4 model group got yellow, and the activity and cibation reduced . The status of prevention group and treatment group were better than that of CCL4 model group.Weight: The weight of nomal group increased obviously, the next in orderwas the prevention group, the treatment group. The CCL4 model group increasedslowly. There is significant difference between model group and normal group （P<0. 01）. The weight of prevention group and treatment group increased to someextent. There is significant difference , compared with model group （ P <0.05）.The index of liver and spleen: The liver index of treatment group and model group are significantly different from that of normal group （ P < 0. 01 ）. The spleen index of prevention group, treatment group and model group are significantly different from that of normal group（ P <0. 01）. The liver index of prevention group is significantly different from that of model group （ P < 0. 01. ） So is the spleen index. There is significant difference between treatment group and model group regarding the liver index.HE stain: The results of pathological grade were analyzed by Ridit method. There is significant difference when the prevention group and treatment group were compared with model group（ P <0.05） . There is significant difference between normal group and model group （ P <0.01）.Hyp and HA in the hepatic tissue: Hyp and HA of prevention group, treatment group and model group are significantly different from normal group （ P <0. 01）. There is significant difference between normal group and prevention group or treatment group （ P < 0. 01）. There is significant difference between modelgroup and other three group （ P < 0. 01）. There is no significant difference between prevention group and treatment group（P >0.05） .S - P immunohistochemistry stain and Western - Blot of the hepatic tissue: Buffy represents positive result. The positive mass increased gradually in the order of the normal group, prevention group, treatment group and model group when observed in the microscope. The data from S - P immunohistochemistry are quantitied by microscope image analysis program. TGFj3i of prevention group, treatment group and model group is significantly different from normal group （ P<0. 01）. So is Smad. TGFPjand Smad2/3 of prevention group and treatment group are significantly different from model group（ P <0. 01）. Regarding TGF(3j there is significant difference between prevention group and treatment group （P<0. 05）TGFp,and Smad2/3 Grey electrophoresis strip of TGFPj and Smad2/3 emerged at 25KD,75KD respectively in Western - Blot. The color becomes deeper and the strap becomes wider gradually from the normal group, the prevention group, the treatment group to the model group. The result by GIS - 2020 gel picture analysis system through protein shifting to NC film was:TGFf$,of prevention group, treatment group and model group is significantly different from normal group（P <0. 01）. So is Smad2/3. TGF(3jand Smad2/3 of prevention group and treatment group are significantly different from model group（ P <0.01） . Regarding Smad2/3 prevention group and treatment group are significantly different from normal group（ P <0. 01） . There is significant difference between prevention group and model group（ P < 0.01）.ConclusionsThis paper discusses the effects of traditional Chinese medicine RuanGan-Ning on the rat model of hepatic fibrosis which was induced by CCL4.The study discovers;Traditional Chinese medicine RuanGanNing can decrease obviously the index of liver and spleen of rats "hepatic fibrosis. Chinese drug RuanGanning can decrease HypNHA concentration in hepatic tissue of rats. Conclusion from HE^S - P and Western - Blot : Traditional Chinese medicine RuanGanning can de-cease the range and degree of hepatic fibrosis. The mechanism may be associated with the inhibition of the expression of TGF(3, NSmad2/3.更多还原