【作者】 郭争荣；

【导师】 李兵顺； 孙殿兴；

【作者基本信息】 河北医科大学 ， 内科学， 2007， 硕士

【摘要】 目的:Ⅰ、Ⅲ型胶原在肝细胞外过度沉积是肝纤维化、肝硬化形成的一个重要特征。然而,到目前为止仍没有一种有效地治愈肝纤维化、肝硬化的方法。中性粒细胞胶原酶(MMP-8)能够特异的降解Ⅰ型胶原,把MMP-8基因导入肝细胞将有可能是降解胶原纤维有效的方法。本研究通过构建一种能够表达MMP-8的腺病毒,治疗肝硬化的大鼠,探讨其能否有效地降解胶原,减轻肝脏的纤维化程度。方法:以质粒pGW1GH-MMP8为模板,应用聚合酶链反应(polymerase chain reaction,PCR)和酶切重组技术克隆了全长的MMP-8基因(pro-MMP8)及其截短的MMP-8基因(tMMP-8)片段,构建了复制缺损性腺病毒载体携带乙肝病毒(HBV)载体表达MMP-8基因的质粒pDC-CH-tMMP8、pDC-C-MMP8,经酶切鉴定正确后进行质粒的大量提取,并用同样的方法制备对照质粒pDC-CH-RFP2,构建的质粒与腺病毒骨架质粒pBHGLox△E1,3Cre共转染293细胞以包装、扩增重组腺病毒。在动物实验中,以3×1011病毒颗粒尾静脉注射治疗硫代乙酰胺诱导的肝硬化大鼠模型,分别于2周、4周后处死动物,0.3%戊巴比妥腹腔注射麻醉,腹主动脉取血,收集血清保存于-70℃,做肝功能、肝纤四项等检查,分离肝组织,部分用10%的福尔马林固定,进行组织病理学检查,其余-70℃保存备用。结果:成功构建了表达MMP-8基因的腺病毒Ad-CH-tMMP8、Ad-C-MMP8,并运用于治疗肝硬化大鼠的研究。在Ad-CH-tMMP8、Ad-C-MMP8尾静脉注射肝硬化大鼠的体内转染研究中,转染2周后相比于模型组和阴性对照组,肝纤维化程度减轻。HE染色及天狼猩红染色结果显示治疗组肝细胞脂肪变性、肝细胞坏死及炎症细胞浸润均有减轻,可见不同程度的肝细胞再生,胶原纤维条索变细变窄,肝小叶结构恢复,与模型组及阴性对照组相比有统计学意义(P<0.05);羟脯氨酸含量测定结果显示与模型组相比,治疗组肝组织羟脯氨酸含量有所下降(28.97±2.36 VS 17.04±0.61、17.62±1.30),具有统计学上的差异(P<0.05),而Ad-CH-RFP2组较模型组又有所升高(30.89±3.67);血清ALT测定结果显示治疗组、阴性对照组大鼠ALT升高,显著高于正常组和模型组(P<0.05),然而,与正常组、模型组、阴性对照组相比,治疗组血清总胆红素、血清白蛋白没有显著的变化,无统计学差异(P>0.05)。转染4周,治疗组大鼠肝纤维化程度继续减轻。而在Ad-CH-RFP2对照组,纤维化程度同模型组类似,并未减轻。结论:实验研究结果表明,MMP-8能够有效地降解Ⅰ型胶原,减轻大鼠肝纤维化程度。这一创新,有望把MMP-8基因治疗肝硬化带入临床应用,开辟肝硬化软肝治疗新途径。更多还原

【Abstract】 Objective:Excess typeⅠ,Ⅲcollagen accumulation is a major feature of liver fibrosis and cirrhosis, reversion of this disease has not been fully accomplished. Neutrophil collagenase(MMP-8) can degrade typeⅠcollagen effectively. Introduction of MMP-8 gene into liver cells could be an advantageous tool to degrade collagen. In this study, we constructed a adenovirus expressing MMP-8 and intended to investigate which can degrade collagens and attenuate established hepatic fibrosis in the rat.Methods:We cloned full length MMP-8 gene (pro-MMP8) and truncated MMP-8 gene (tMMP-8) from plasmid pGW1GH-MMP8 by polymerase chain reaction(PCR) and enzyme-cutting recombination technique, and constructed the plas- mid pDC-CH-tMMP8, pDC-C-MMP8 which express MMP-8 in adenoviral vector carrying replication-deficient HBV vector , we have also constructed the control plasmid pDC-CH-RFP2. The constructed plasmids were conformed by enzyme-cutting technique and then co-transformed with pBHG1ox△E1,3Cre into 293 cells in order to package and expand recombinant adenovirus. Liver cirrhosis rats induced by TAA for ten weeks were infected once with recombinant adenovirus(3×1011 viral particles per kilogram) by intravenous injection through tail veins. At 2 or 4 weeks after the adenoviral infection, rats were killed. Animals were injected with 0.3% Pentobarbital and killed thirty minutes later. Blood samples were collected by puncture of the abdominal aorta, and serum was stored at -70℃until analysis.Samples of the liver were snap-frozen at -70℃or were fixed in 10%buffered formalin for subsequent histological analysis.Results:We have successfully constructed the recombinant adenovirus expressing MMP-8(Ad-CH-tMMP8, Ad-C-MMP8) and test the activity of MMP-8 in animal exprement, compared with model group and negative control group, the fibrosis was dramatically attenuated at two weeks after the infection, Compared with model group and negative control group,HE staining and bitter acid-sirius red staining showed that hepatocyte steatosis, necrosis and inflammation were significantly mild in treatment group, along with hepatocyte proliferation and recovery of hepatic lobules structure(P<0.05); compared with model group, the content of HYP diminished(28.97±2.36 VS 17.04±0.61, 17.62±1.30; P<0.05);compared with model group and normal group, measurement of serum ALT showed that adenovirus expression in the liver transiently induced a significant increase in serum ALT(P<0.05). However, compared with normal group、model group and negative control group, serum total bilirubin and serum albumin levels did not change significantly in treantment group(P>0.05). After 4 weeks, the proliferative effect almost disappeared, but the hepatic fibrosis remained attenuated and the hydroxyproline content remained diminished. Whereas, the fibrosis in Ad- CH-RFP2-treated rats persisted.Conclusion:Studying results shows that the MMP-8 gene can degrade typeⅠcollagen effectively and attenuate established fibrosis in rat induced by TAA. It is promising for use in a clinical setting and providing a novel therapy tool for liver fibrosis.更多还原