【作者】 程良斌；

【导师】 张赤志；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2006， 博士

【摘要】 黛矾散对雌激素诱导肝内胆汁淤积大鼠肝细胞膜影响的实验研究目的 肝内胆汁淤积（intrahaptic cholistasis，IC）是许多肝病的共有的基础病变，其病因包括感染、药物、妊娠、酒精、自身免疫、手术、遗传等多种因素。近年来，随着口服避孕药及雌激素替代疗法的应用，雌激素诱导的肝内胆汁淤积发病率有增高的趋势。国外对肝内胆汁淤积症发病机制的研究已深入分子水平，但在治疗方面，除最近推崇的胆石溶石药熊去氧胆酸（UDCA）外，尚缺乏有效的治疗药物。我国有关肝内胆汁淤积的研究正处于起步阶段，西药研究滞后，而中药治疗已初见成效，并显现出一定的优势和特色。但有关中药治疗方面的研究多为临床研究，且多限于改善症状的观察，缺乏确切的科学实验依据。临床观察证实，黛矾散对病毒性肝炎、胆道疾患、妊娠等引起的胆汁淤积有较好疗效，但缺乏实验印证。基于上述国内外研究现状，我们以苯甲酸雌二醇（EB）诱导的肝内胆汁淤积模型为基础，用拆方研究方法，观察中药黛矾散对肝内胆汁淤积模型大鼠肝功能、肝组织学及肝细胞超微结构变化、肝细胞膜流动性、肝匀浆及肝窦膜中Na⁺-K⁺-ATPase、Mg⁺⁺-ATPase活性的影响，探讨黛矾散治疗肝内胆汁淤的药物作用靶点和可能作用机制，从而为中医药治疗肝内胆汁淤积症提供科学依据，为寻求治疗肝内胆汁淤积的有效药物研究打下基础。 方法 1、雄性Wistar大白鼠30只，体重280-320g，随机分为5组，每组6只，分别为正常组（A）、模型组（B）、明矾组（C）、青黛组（D）及黛矾散组（E）。B、C、D、E等四组分别以EB橄榄油皮下注射（11mg／kg.d），连续3d进行造成肝内胆汁淤积模型。 2、观察实验动物小便颜色、毛色、饮食量和活动情况；取各组大鼠肝脏，用4％甲醛常规固定，制成石腊切片，进行HE常规染色，光学显微镜下观察肝脏取肝组织学变化；超薄切片机切片，醋酸双氧铀、枸橼酸铅双重染色，透视电子显微镜观察肝细胞超微结构变化。 3、分离血清，常规生化检测各组实验大鼠血清总胆汁酸（TBA）、总胆红素（TBiL）、ALT、ALP、总胆固醇（Tch）的变化。 4、采用DPH荧光探针技术，用荧光偏振度测定及微黏度的计算的方法，测定肝细胞亚膜（窦膜和毛细胆管膜）流动性的变化。 5、取肝组织进行匀浆，分离毛细胆管膜和肝细胞窦膜，分别检测肝匀浆和毛细胆管膜中ALP水平，检测肝匀浆及肝窦膜中Na⁺-K⁺-ATPase、Mg⁺⁺-ATPase活性。 结果 1、一般观察可见，经EB皮下注射，模型组动物小便污染的白色草纸颜色黄更多还原

【Abstract】 Objective Intrahepatic cholestasis（IC） is the common fundational pathologic changes. Recently the sick rat of IC is rising caused by estrogen with more and more wemen taking estrogen. The study in pathogenic mechanism of IC have penetrated into the molecular level in forenge, but there was not any effective treatment pharmaceuticals besides ursodeoxy cholic acide（UDCA）. In my country, the study of IC is in the class of lower primary but the Chinese medicine has showed the primary effective and some superiority and speciality.The main study in Chinese medicine to IC focused at clinical and was limited to observating changes of clinical symptoms but was short of the definite experiment’s foundation. The clinical studies proved that Chinese medicine Dai Fan San（made up by Ming Fan and Qing Dai） was a sort of effective treatment to IC caused by virus hepatitis ,deseases of biliary tract and pregnancy et al, but it was short of the experiment’s foundation. In accordance with the progress, the aim of our study was to probe into the pharmaccoligy mechanism of Dai Fan San and supply the science basis and establish the base of seeking for active material medica to IC ,through observing the effect of Dai Fan San and its components to rat’s liver function , liver histology, liver cell ultrastructure, hypatocyte memberane fluidity and Na⁺-K⁺-ATPase, Mg⁺⁺-ATPase of IC induced by estradio benzate（EB）. Methods Use EB to replicate cholestasis model and observe the effect of Dai Fan San and its components on the liver function （TBA,Tbil,ALT and ALP）, liver histology, liver cell ultrastructure, hypatocyte sub-membrane fluidity（cluding hypoctocyte sinus membrane and bile capillaries membrane ） and Na⁺-K⁺-ATPase、 Mg⁺⁺-ATPase in liver homogenate and hypoctocyte sinus membrane of rat’s IC induced by EB.Results The results of the experiment show that the rat’s liver function （TBA,Tbil,ALT and ALP） all go up distinctly （PO.05） ,but Tch go down obviously （P<0.05）; ALP in hepatic even cytoplasm and cholangiole membrane go up prominenty （P<0.05）; the activity of Na⁺-K⁺-ATPase、 Mg⁺⁺-ATPase all go down obviously （P<0.05）; Dai Fan San and its components filled by mouth.can always hider更多还原