【作者】 张红磊；

【导师】 李佃贵；

【作者基本信息】 河北大学 ， 中西医结合临床， 2010， 硕士

【摘要】 目的:本实验通过无菌未灭活的猪血清腹腔注射的方法制备肝纤维化大鼠模型,探讨化浊解毒调肝方抗大鼠肝纤维化的作用及其可能的机制,为肝纤维化的治疗提供理论及实验依据。主要内容包括:观察化浊解毒调肝方对肝纤维化大鼠肝组织病理形态学的影响;化浊解毒调肝方对肝纤维化大鼠肝组织TGFβII型受体(TβRII)mRNA表达的影响;化浊解毒调肝方对肝纤维化大鼠肝组织smad4蛋白表达的影响。方法:清洁级Wistar大鼠50只,雄性,体重200-220g。适应性喂养一周后随机抽取10只作为正常组。造模成功后将剩余40只大鼠随机分为:模型对照组、秋水仙碱组(简称西药治疗组)、化浊解毒调肝方高剂量组(HZG)、化浊解毒调肝方低剂量组(HZD),每组10只。各组均普通颗粒饲料喂养,自由饮水和进食,分笼饲养于20±2℃,湿度65%±5%清洁级动物实验室内。除正常对照组外,其余40只均采用未灭活的猪血清腹腔注射,每次0.5ml/只,固定时间为周一、周五上午9点,每周进行两次,连续8周。秋水仙碱治疗组和中药治疗组于造模的第8周末给药,化浊解毒调肝方低剂量组给予含1.5g/ml生药水煎剂灌胃;化浊解毒调肝方高剂量组给予含3.0g/ml生药水煎剂灌胃;秋水仙碱组给予0.154g/kg·d灌胃,均至12周末。每天上午灌胃一次,每次3ml,连续4周。正常对照组与模型组给予等量生理盐水灌胃。于第12周末处死大鼠,留取大鼠肝组织,采用苏木精-伊红染色和Masson染色(胶原纤维特殊染色法)观察肝组织病理形态学的变化。采用RT-PCR技术测定肝组织TGFβII型受体(以下简称TβRII ) mRNA表达情况,采用免疫印迹法(Western-immunoblotting)测定大鼠肝组织中smad4蛋白的表达,所得数据经过整理,数据用均数±标准差表示,采用SPSS13.0统计软件进行处理。两个样本均数比较采用t检验,多个样本均数比较行完全随机设计单因素方差分析。结果:1.一般情况:正常对照组大鼠食欲佳,被毛光滑,精神状态好,体重增加。模型组大鼠精神萎靡,活动减少,食欲减退,消瘦,皮毛无光泽,体重较前有所下降,进食较正常组少、尿黄,有的大鼠鼻有出血。西药治疗组及中药治疗各组大鼠精神、食欲及活动介于正常组与模型组之间。2.化浊解毒调肝方对肝纤维化大鼠肝脏病理形态学的影响肉眼观察,正常组大鼠肝脏质地软,颜色呈深红色,表面光滑,边缘锐圆。模型组大鼠肝脏明显肿大,质地较硬,颜色片偏暗红,表面有结节形成而粗糙不平,有的可见颗粒样凸起,边缘较纯。秋水仙碱组和中药治疗各组大鼠肝脏肝脏肿大不明显,表面比较光滑,偶见沙粒样增生,颜色略见暗红,质地较脆。光镜下观察,正常组肝小叶结构完整,肝细胞围绕中央静脉呈放射状的排列,肝细胞核大而圆,无变性坏死,未见明显的纤维结缔组织增生。模型组可见肝小叶结构被严重破坏,肝细胞广泛的脂肪变性、坏死并伴炎性细胞浸润,纤维组织大量增生,个别动物可见假小叶形成。中药治疗各组和西药对照组肝组织病理的损伤程度与模型组相比均可见有不同程度的修复,尤其是化浊解毒调肝方高剂量组,肝细胞脂肪变性及炎性细胞浸润较模型组减少,胶原纤维增生较轻,主要散在于肝血窦和disse间隙、汇管区,无假小叶形成。3.化浊解毒调肝方对肝纤维化大鼠肝组织TβRIImRNA表达的影响与正常对照组相比,模型对照组大鼠肝组织TβRIImRNA的表达明显增强(P<0.01);化浊解毒调肝方高剂量组、低剂量组及秋水仙碱组与模型组相比TβRIImRNA的表达均有明显下降(P<0.01);化浊解毒调肝方高剂量组与秋水仙碱组相比TβRIImRNA的表达有显著性降低(P<0.01);化浊解毒调肝方高剂量组与化浊解毒调肝方小剂量相比TβRIImRNA的表达有显著性降低(P<0.01);化浊解毒调肝方小剂量组与秋水仙碱组相比TβRIImRNA的表达无明显差异(P>0.05)。4.化浊解毒调肝方对肝纤维化大鼠肝组织smad4蛋白表达的影响与正常对照组相比,模型对照组大鼠肝组织smad4蛋白的表达量明显增强;化浊解毒调肝方高剂量组、低剂量组及秋水仙碱组与模型组相比smad4蛋白的表达量均有明显下降(P<0.01);化浊解毒调肝方高剂量组与秋水仙碱组相比smad4蛋白的表达量有显著性降低(P<0.01);化浊解毒调肝方高剂量组与化浊解毒调肝方小剂量相比smad4蛋白的表达量有显著性降低(P<0.01);化浊解毒调肝方小剂量组与秋水仙碱组相比smad4蛋白的表达量无明显差异(P>0.05)。结论:1.化浊解毒调肝方能有效改善肝纤维化大鼠肝组织病理改变、减轻肝细胞的损伤,减少胶原纤维的生成,从而发挥其抗纤维化的作用。2.化浊解毒调肝方能显著降低大鼠肝组织TGF-βII型受体(TβRII)mRNA和smad4蛋白的表达,抑制肝星状细胞的活化,通过调控相关细胞因子生物作用,促进细胞外基质的降解。因此,以浊毒立论治疗肝纤维化具有实用性和可行性。更多还原

【Abstract】 Objectives : This experiment aims to explore the effect of HuazhuoJiedu TiaoGan’formula on pig’s serum -induced Liver Fibrosis in rats. Investigate on Pathological Mechanism of Hepatic fibrosis,so that provide theoretical and experimental basis for treating liver fibrosis.The research included HuazhuoJiedu TiaoGan’formula on the expression of TβRII mRNA in liver tissue of hepatic fibrosis rats; HuazhuoJiedu TiaoGan’formula on the expression of Smad4 in liver tissue of hepatic fibrosis rats.Methods: Total 50 clean male Wistar rats weighed 200～220g, normal diet a week after acclimation then randomly selected 10 cases as the normal group (normal diet, administered three times a day, a total of 12w).After the success of the model the rats were randomly divided into four groups: model group (MD), colchicine Group(CG),HuazhuoJiedu TiaoGan’formula high dose group (HZG), HuazhuoJiedu TiaoGan’formula of low-dose group (HZD). In addition to the normal group,pig’s serum( 0.5ml per mouse, twice a week) was established in rats by intraperitoneal injection for eight weeks. The treatment groups were given drug at the end of the eighth week. The rats in the normal control and model groups received intragastric administration of saline(3ml each time).At the 12th weekend,the specimens were taken and the degree of hepatic fibrosis was judged by routine haematoxylin-eosin staining and Masson staining. TGFβ1 II receptor mRNA expression were determined using RT-PCR technique. Smad4 protein expression were determined using Western-immunoblotting technique. Data was collected and analyzed through t test or analysis of variance. The statistic software was SPSS13.0. Differences with P values <0.01 were considered significant.Results1 General performance:normal control rats, loss of appetite good, hair smooth, good mental state, weight gain. Model rats make them apathetic, reduced activity, anorexia, weight loss, dull coat, weight over the previous decline. Treatment of rats in each group spirit, appetite and activity between the normal group and model groups. 2 Effect of HuazhuoJiedu TiaoGan’formula to pathomorphology in hepatic fibrosis rats.The structure of hepatic lobule were integrated hepatic cord radially ranged as the center of navel vein.There was not degeneration and necrosis in liver tissue. There was not collagen proliferation.Compared with normal group,the model control rats had obvious liver inflammation including degeneration, necrosis, fibrosis and collagen proliferation. Pseudolobule proliferation could be seen in individual animal. At the end of 12th weeks, Pathological examination in the treatment groups less injured than in model group. compared to model control group,Chinese medicine and western medicine treatment group have varying degrees of improvement and recovery.3 Experimental study on HuazhuoJiedu TiaoGan’formula effect on TGFβ1 II receptor mRNA of the immunity hepatic fibrosis rats.The results showed that the expressions of TβRIImRNA in the model groups were increased significantly, respectively compared with normal control group and the difference was significant (P<0.01). Compared with the model group, all medicine intervention group decreased the expressions of TβRII mRNA of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.01). HuazhuoJiedu TiaoGan’formula high-dose group and the low dose groups were significantly different (P <0.01);HuazhuoJiedu TiaoGan’formula high-dose group and Colchicine group were significantly different (P <0.01);Western medicine group and HuazhuoJiedu TiaoGan’formula low-dose groups had no significant difference (P> 0.05);4 Experimental study on HuazhuoJiedu TiaoGan’formula effect on smad4 of the immunity hepatic fibrosis rats.The results showed that the expressions of smad4 in the model groups were increased significantly, respectively compared with normal control group and the difference was significant (P<0.01);Compared with the model group, all medicine intervention group decreased the expressions of smad4 of immunity hepatic fibrosis rats at the end of 12 weeks,There were statistic differences among the groups(P<0.01);. HuazhuoJiedu TiaoGan’formula high-dose group and the low dose groups were significantly different (P <0.01); HuazhuoJiedu TiaoGan’formula high-dose group and Colchicine group were significantly different (P <0.01);Western medicine group and HuazhuoJiedu TiaoGan’formula low-dose groups had no significant difference (P> 0.05);Conclusions1 HuazhuoJiedu TiaoGan’formula can improve the liver pathologic injur and ameliorate hepatocyte injury in hepatic fibrosis rats , exert an anti-hepatic fibrosis effect.2 HuazhuoJiedu TiaoGan’formula could reduce the expression of TβRII mRNA and smad4 of immunity hepatic fibrosis rats, suppresse theaetivation of HSCs, decrease the deposition of ECM. therefore, involving Huazhuo Jiedu in the treatment of hepatic fibrosis has practicability and feasibility.更多还原