【作者】 李玉莲；

【导师】 范红； 宋正己；

【作者基本信息】 昆明医科大学 ， 内科学， 2012， 硕士

【摘要】 第一部分：龙血竭对药物诱导大鼠肝纤维化的干预作用及可能机制目的：观察龙血竭对药物诱导的肝纤维化大鼠的治疗作用并初步探讨其作用机制。方法：健康清洁级SD雄性大鼠36只,体重(200±20)g,随机分为3组：正常对照组、模型组、药物干预组,每组12只,以硫代乙酰胺诱导大鼠肝纤维化。模型组、药物干预组均给予10%硫代乙酰胺-生理盐水溶液腹腔注射(首次250mg/kg,此后200mg/kg),空白组给予生理盐水0.5m1腹腔注射,2次/周。2周后,模型组、空白组每天加用生理盐水2ml灌胃。药物干预组：每只每天加用龙血竭0.6克溶于3ml生理盐水灌胃,共8周。第8周末处死大鼠,测定各组肝纤维化大鼠血清中谷丙转氨酶、谷草转氨酶及透明质酸酶、层粘连蛋白、IV型胶原的含量。取肝组织作病理切片,进行HE染色观察肝脏的病理变化,masson染色观察肝组织中胶原的增生程度。结果：硫代乙酰胺8周后成功诱导大鼠肝纤维化模型。模型组大鼠血清中透明质酸酶、层粘连蛋白和IV型胶原水平较正常组均升高(P<0.05),有统计学意义；给药六周后,大鼠血清透明质酸酶、层粘连蛋白和IV型胶原水平与模型组相比明显降低(P<0.01)；模型组大鼠血清中谷丙转氨酶和谷草转氨酶水平较正常组均显著升高(P<0.05),药物干预组大鼠血清谷丙转氨酶和谷草转氨酶水平与模型组相比明显降低(P<0.01)；纤维化程度：正常对照组大鼠无肝纤维化；模型组与正常对照组大鼠相比肝脏组织纤维化程度更高,药物肝脏纤维化程度均优于模型组；masson染色后观察,模型组胶原纤维沉积较为明显,药物干预组胶原纤维沉积较模型组有明显改善。结论：硫代乙酰胺可成功诱导大鼠肝纤维化。龙血竭能够降低肝纤维化大鼠血清中透明质酸酶、层粘连蛋白和IV型胶原及谷丙转氨酶和谷草转氨酶水平,并能改善纤维化大鼠程肝脏的病理损伤程度,减少肝纤维化大鼠肝组织中胶原纤维的增生,上述作用可能与其主要成分龙血素B单体抑制肝星状细胞的增殖,促进活化的肝星状细胞凋亡,从而抑制参与肝纤维化及血管新生启动与进展的重要因子有关。第二部分：龙血素B对肝星状细胞增殖及促血管新生的影响目的：观察龙血素B对肝星状细胞增殖及透明质酸酶、层粘连蛋白、IV型胶原含量的影响。检测肝星状细胞药物处理前后血管内皮生长因子、低氧诱导因子、神经纤毛蛋白1mRNA表达的影响。方法：用MTT法观察龙血素B对肝星状细胞增殖的影响。计算IC50,以IC50,IC50/2, IC50/10三个浓度处理细胞,并检测细胞上清中透明质酸酶、层粘连蛋白、Ⅳ型胶原的含量；采用荧光实时定量PCR(RT-PCR)(?),检测肝星状细胞药物处理前后血管内皮生长因子、低氧诱导因子、神经纤毛蛋白1mRNA表达的影响。结果：龙血素B的IC50为0.3ug.ul-1;与正常对照组相比药物干预组细胞上清中透明质酸酶、层粘连蛋白和Ⅳ型胶原水平均减低(P<0.05)；与正常对照组相比药物干预组细胞内VEGF和HIF-1及NRP-1的基因表达明显下调。结论：龙血素B可抑制肝星状细胞的增殖,并降低细胞上清中透明质酸酶、层粘连蛋白和Ⅳ型胶原的含量,还可下调细胞内血管内皮生长因子,神经纤毛蛋白1和低氧诱导因子mRNA的表达。更多还原

【Abstract】 Part I:Loureirin B on experimental hepatic fibrosis and its possible mechanismObjective:To investigate Loureirin B on experimental hepatic fibrosis, and present possible mechanisms。Method:36clean male SD (Sprague-Dawley) rats were randomly divided into4groups:Control group, model group, drug treatment group; With thioacetamide (TAA) inducing liver fibrosis model, to bserve serum transaminase (ALT, AST), fibrosis markers (HA, LN, IV-C), liver histopathology changes.Results:To compare with model group,(1)liver pathology grade, Control group were more serious than the model group; drug treatment group were better than the model group;(2) The level of drug treatment group ALT, AST decreases significantly (P<0.01),(3) The level of drug treatment group HA, LN, IV-C is significantly decreased (P<0.01)。Conclusion:Rat liver fibrosis model induced by TAA is established Successfully; dragon’s blood can protect TAA-induced liver fibrosis in liver function, and improve the quality of life in rats with liver fibrosis; dragon’s blood treatment on liver fibrosis may be positively correlated with the dosage. Part Ⅱ:Effect of Loureirin B on Proliferation and Extracellular Matrix Secretion of Rat Hepatic Stellate Cells in Vitro Objective:To investigate the effects of loureirin B on proliferation and extracellular matrix secretion of rat hepatic stellate cells in vitro.Methods:HSC-T6was cultured in96-well plates for24hours.Then they were incubated with different concentration of loureirin B for48hours.MTT colorimetry was used for assaying proliferation of hepatic stellate cells.lnhibition rate was calculated. Hyaluronic acid Laminin and Collagen type IV were measured by radioimmunoassay.The expression of vascular endothelial growth factor hypoxia-inducible factor and neuropilin-1mRNA.Results:Addition of loureirin B into culture medium could significantly inhibit HSC proliferation,and the higher concentration of the loureirin B the stronger inhibition rate of the hepatic stellate cells. It also inhibit Hyaluronic acid、Laminin and Collagen type IV secretion in different degree. The expression of vascular endothelial growth factor hypoxia-inducible factor and neuropilin-1mRNA.Conclusion:Loureirin B can significantly inhibite hepatic stellate cells proliferation and extracellular matrix secretion. And inhibite the expression of vascular endothelial growth factor hypoxia-inducible factor and neuropilin-1.更多还原