【作者】 陈泓；

【导师】 贾玉杰；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的:观察肝复康作用实验性肝纤维化大鼠后血清细胞外基质标志物(Ⅳ型胶原、Ⅲ型前胶原、透明质酸、层粘连蛋白)的变化情况及与细胞外基质降解与沉积有关的基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶抑制因子-2(TIMP-2)基因表达变化。以期深入探讨肝纤维化的发生、发展及逆转过程机制,最终为临床诊断及改善肝纤维化的治疗现状提供新思路。方法:健康SD大鼠77只,将其随机分为正常对照组(C)10只,模型组(M)11只,模型对照组(MC)11只,低剂量治疗组(LT)10只、中剂量治疗组(MT)12只、高剂量治疗组(HT)12只,预防组(P)11只。除正常对照组外,余鼠造模。皮下注射以橄榄油稀释的10%的四氯化碳,5ml/kg,每周两次,共12周,正常对照组以同样方法皮下注射生理盐水。其中预防组于造模同时以312.5 mg /kg﹒d剂量的肝复康煎剂(主要成分为丹参、黄芪、赤芍、白芍、柴胡等)灌胃12周。高剂量治疗组、中剂量治疗组、低剂量治疗组则于造模第9周后开始分别以3125mg/kg﹒d、312.5mg/kg﹒d、31.25mg/kg﹒d剂量的肝复康灌胃12周。以上各剂量药物均溶于10 ml/kg的生理盐水中。正常对照组及模型对照组则以等容量的生理盐水灌胃。第12周末将模型组、预防组大鼠隔夜禁食,次日称量体重后,以乙醚麻醉腹主动脉采血检测各项指标,处死大鼠,迅速取出肝组织,从肝右叶的中部切取肝组织于-70℃冰箱保存用于RT-PCR。第20周末将其余各组大鼠以同样方法处死,取出肝组织,备用。应用放射免疫分析法检测大鼠血清样本中各项指标的变化情况(Ⅳ型胶原、Ⅲ型前胶原、透明质酸、层粘连蛋白);采用RT-PCR法检测各组大鼠肝组织中基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶抑制因子-2(TIMP-2)基因的表达情况,用Olympus显微摄像系统采集图片,用图像分析软件测定其积分光密度(IOD),以IOD值表示表达水平,对IOD值进行分析和比较。结果:1、模型组及模型对照组血清Ⅳ型胶原、Ⅲ型前胶原、透明质酸、层粘连蛋白含量较之正常对照组均有明显增高(P=0.000),预防组与正常对照组血清四项指标比较无显著差异(P>0.05)。经不同剂量的中药肝复康干预后,该四项指标血清值均有明显降低(P<0.05),并以中剂量组作用效果最明显;2、基于RT-PCR和其IOD值检测得到的结果提示:MMP-2和TIMP-2在正常对照组表达微弱,在肝脏纤维化模型及模型对照组表达较正常对照组显著增强(P<0.05),经不同剂量的中药肝复康治疗后表达均有明显降低(P<0.05),其中中剂量治疗组降低水平最为显著,其表达水平基本恢复至正常大鼠相应表达水平,治疗效果优于高剂量、低剂量。结论:①中药肝复康具有较好的预防和治疗肝纤维化作用,并且以中剂量作用效果最为明显,是较为合理的用药剂量。②中药肝复康可以通过抑制胶原合成,减少ECM生成减轻大鼠肝纤维化程度。③中药肝复康可能通过解除MMP-2的抑制因素而提高其活性,以促进胶原降解。④中药肝复康通过调控基因表达水平,抑制MMP-2和TIMP-2mRNA表达,使MMP-2/TIMP-2趋于平衡,从而发挥其抗纤维化的作用。更多还原

【Abstract】 Objective To observe the changes of serum HA,LN,CIV,PCⅢand MMP-2、TIMP-2 which are correlated with the extracellular matrix (ECM) degradation after the effect of GFK on experimental rats with hepatic fibrosis. To elucidate the expression patterns of MMP-2, TIMP-2. To determin the potential genetic mechanism of hepatic fibrosis’s happen, development and reverse. To provide an adjuvant therapeutic clue for better treatment of human hepatic fibrosis.Methods Healthy SD rats were divided into seven groups at random including normal control group(n=10),Ganfukang high dose group(n=12),Ganfukang medium dose group (n=12),Ganfukang low dose group(n=10),model group(n=11), model control group(n=11), and preventive group (n=11). All groups except normal control group were subcutaneous injected 10% CCl4(5ml/kg) for 12 weeks and two times each week to obtain hepatic fibrosis and were treated with sodium chloride,GFK3125mg/kg ,GFK312.5mg/kg, GFK31.25mg/kg respectively after nine weeks of CCl4 injection. Model control group were treated with sodium chloride. Preventive group were treated with GFK 312.5mg/kg.d for two week as soon as the model is established. At the end of the twelfth week, rats in model and preventive groups were put to death, reserve the serum to detect four ECM targets. Also fetch out the liver as quickly as possible and reserve in the -70℃refrigeratory for the RT-PCR. The mRNA expression of MMP-2 and TIMP-2 were measured by semi-quantitation reverse transcriptase polymerase chain reaction (RT-PCR). The RT-PCR results was input into the image analysis software to detect the integrated option density(IOD)to show expression level and so to analysis and compare. At the end of the twenties week, the left rats were treated with the same methods as the above. RIA technique were applied to detect the serum levels of CIV,PCⅢ,HA,LN.Results 1) The serum level of HA,LN,CIV,PCⅢwere higher in the model and model control groups, which has significant difference compare with normal control group (P<0·05). There is no significant difference between preventive and normal control group (P>0·05). The collagen fibrosis decreased significantly in GFK group, this groups has significant difference compare to model group (P<0·05). And the effect of medium dose GFK is the most effective. 2) RT-PCR demonstrated that expression of MMP-2 and TIMP-2 in model group increased significantly compared with normal control group,While the expression of MMP-2 and TIMP-2mRNA in the therapeutic groups reduced significantly compared with model group. The same result we can get is that the effect of medium dose GFK is the most effective.Conclusion 1) GFK has preferable preventive and therapy effects on rats with hepatic fibrosis. And the effect of medium dose GFK is the most effective. It’s the reasonable dosage.2) GFK can significantly relieve the degree of hepatic fibrosis. This may be through the way that GFK inhibit synthesis of collagen and reduce generation of ECM. 3) GFK can probably remove the MMP-2 inhabitor so to improve the bioactivity of MMP-2 to promote the ECM degradation. 4) GFK may play its anti-fibrosis function through regulates gene expression, repress the expression of MMP-2 and TIMP-2 mRNA to make MMP-2 and TIMP-2 tend to be balanced.更多还原