【作者】 贺欣；

【导师】 艾群；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2009， 硕士

【摘要】 目的:在正常肝脏的纤维组织中存在着细胞外基质(ECM)生成与降解的动态平衡,肝纤维化是ECM生成与降解过程失衡的结果[1,2]。既往对ECM的生成与沉积研究较多,近年开始对ECM降解的研究也日益深入,但是在肝纤维化临床治疗领域,并没有取得明显的进步。中药肝复康是本校病理生理教研室多年来通过反复实验总结出的抗纤维化经验方,本试验拟从ECM降解的角度探讨肝复康的作用机制。方法:将SD大鼠随机分为正常对照组(C)、模型组(M)、模型对照组(MC)、高剂量治疗组(HT)、中剂量治疗组(MT)、低剂量治疗组(LT)和预防组(P)。除正常对照组外,余鼠用皮下注射四氯化碳的方法复制大鼠肝纤维化模型。处死各组大鼠后测其血清丙氨酸转氨酶( alanine aminotransferase,ALT)、天冬氨酸转氨酶( aspartate- aminotransferase,AST)、白蛋白(albumin,A)、球蛋白(globulin, G)的含量;HE染色法观察肝组织病理学变化;免疫组化检测α-平滑肌肌动蛋白(α-SMA)的表达;逆转录聚合酶链(reverse transcription polymerase- chain reaction, RT-PCR)法观察基质金属蛋白酶抑制剂-1(TIMP-1)、Ш型胶原(CollagenШ)mRNA表达量的变化。同期正常饮食饲养大鼠作对照。结果:1.血清学指标:与模型组比较,肝复康治疗后可明显减少ALT及AST的释放,升高血清白蛋白,降低血清球蛋白,升高白球比,以中剂量治疗组最为显著。2.肝组织病理学变化:与模型组比较,肝复康治疗组肝细胞变性及坏死程度减轻,汇管区及小叶间仅有少量纤维组织增生,纤维间隔细小,尤以中剂量治疗组差异显著。3.肝组织免疫组化染色:模型组α-SMA主要表达于汇管区及纤维间隔,且呈长椭圆形或梭形,免疫组织化学染色面积显著高于正常对照组。中剂量治疗组α-SMA表达较模型组明显下降,与正常对照组无明显差异。4.肝组织TIMP-1和CollagenШmRNA的表达:模型组大鼠肝组织TIMP-1 mRNA表达较正常对照组明显上调。模型对照组、高剂量治疗组、中剂量治疗组和低剂量治疗组大鼠肝组织TIMP-1 mRNA表达较模型组均显著下调,其中以中剂量治疗组最为明显,预防组大鼠肝组织TIMP-1 mRNA表达较模型组没有显著性差异。模型组大鼠肝组织CollagenШmRNA表达较正常对照组明显上调。中剂量治疗组大鼠肝组织CollagenШmRNA表达较模型组明显下调,有显著性差异,且与正常对照组比较无显著性差异。结论:1.肝复康能显著降低CCl4诱导的大鼠肝纤维化的程度,降低TIMP-1及CollagenШmRNA表达水平,对肝纤维化有良好的治疗作用。2.肝复康对CCl4诱导的肝纤维化有疗效,作用机制可能与其下调TIMP-1表达水平,从而减轻了其对MMPs的抑制有关。更多还原

【Abstract】 Objective: There is a dynamic balance between extracellular matrix (ECM)formation and degradation in the fibrous tissue of normal liver. The imbalance between ECM formation and degradation results in hepatic fibrosis. There are more research on the formation and deposition of ECM in the past, while recent years, research on the degradation of ECM are also increasing. However, there have been no tangible progress in the field of clinical treatment of hepatic fibrosis. Developed by pathophysiology depar- tment Ganfukang has been used as the traditional Chinese medicine for treating hepatic fibrosis. Clinical observations in our affiliated hospital have proved its effective. However, further study of its detailed anti-hepatic fibrosis mechanism is still needed. On the basis of the above mentioned theory and research developments, our study was to observe the mechanism of Ganfukang from the perspective of ECM degradation.Method:All SD rats were divided into seven groups: hepatic fibrosis model group(M group)、low dose medicine group(LT group)、middle dose medicine group(MT group)、high dose medicine group(HT group)、model control group(MC group)、medicine prevention group(P group)and normal control group(C group). Rats were treated subcutaneously with CCl4 to produce the model of hepatic fibrosis. Serum alanine aminotransferase (ALT)、aspartate aminotransferase (AST)、albumin(A)、globulin(G)、the ratio of albumin and globulin(A/G) were measured; The liver pathology changes were observed under light microscopy by HE staining; hepatic expression ofα-smooth musle actin(α-SMA) were observed by immunohis- tochemistry; hepatic expression of tissue inhibitor of metalloproteinase -1(TIMP-1) mRNA and Collagen III mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR). At the same period, the rats fed common chow were as normal control.Result: 1.Serum index: Compared to M group, Serum ALT and AST release can be significantly reduced, serum albumin and the ratio of albu- min and globulin can be significantly increased after treating with Ganfu- kang, especially in MT group.2.Pathology of hepatic tissue: The formation of fibrosis, marked fatty degeneration, necrosis, inflammation of hepatocytes, and infiltration of inflammatory cells including macrophages and lymphocytes were seen in the rat livers of M group. Compared to M group, the hepatic necrosis and infiltration of inflammatory cells, particularly in the pericentral region, were significantly diminished in the MT group.3.Immunohistochemistry staining: In the M group, the a-SMA positive cells, representing the transformed HSC, increased in number, size, and immunohistochemical staining intensity compared with those in the other groups.4.Expression of TIMP-1 and Collagen III mRNA: Rats in M group showed higher TIMP-1 levels in liver. The levels decreased in MT group comparison with those in M group, the difference was significant. However, compared with TIMP-1 mRNA in the other groups, there were no significant differences. We found that the pattern of Collagen III mRNA levels was upregulated in M group comparison with those in C group. Compared with Collagen III mRNA expression in M group, the level in MT group decreased significantly.Conclusion: 1.Chinese medicine Ganfukang was a good treatment for liver fibrosis. It significantly reduces the level of TIMP-1 and collagen III mRNA expres- sion in CCl4 -induced rat liver fibrosis.2.The mechanism of Ganfukang’s effection on CCl4-induced liver fibrosis, may be related to reduced expression of TIMP-1 levels, thus reducing their inhibition to MMPs.更多还原