重组人肝再生增强因子反式激活作用的研究

发布时间：2018-05-28 18:41

  本文选题：重组人肝再生增强因子 + 细胞周期蛋白依赖性激酶1　； 参考：《郑州大学》2005年硕士论文

【摘要】：病毒性肝炎及其相关的肝硬化,在我国发病率高、危害严重,严重威胁着人民群众的身体健康,并给病人带来了沉重的经济负担,但目前还没有确切有效的治疗手段。无论是慢性病毒性肝炎,还是肝硬化,都存在不同程度的肝细胞损伤,在病毒性肝炎的综合治疗方案中,促进肝细胞再生占有重要的地位,特别是对于重型肝病。肝再生增强因子(Augmenter if liver regeneration,ALR)在肝细胞的再生过程中发挥着举足轻重的作用。ALR是新近克隆的蛋白质因子,能特异地刺激肝源细胞的增殖,并在动物模型中对急性肝衰竭和慢性肝病有救治作用。但其作用机制尚不清楚,开展ALR作用机制的研究,将为肝病的治疗提供理论依据和新思路。 目的 (1) 应用抑制性削减杂交技术筛选重组人肝再生增强因子(recombinant human aumenter of liver regeneration,rhALR)反式激活的基因,探讨rhALR刺激肝细胞再生的作用机制;(2) 研究重组人肝再生增强因子(rhALR)对细胞周期蛋白依赖性激酶1(cdk1)基因的反式激活作用。 方法 (1) 应用大肠杆菌系统,表达、纯化重组人肝再生增强因子蛋白。体外刺激HepG2细胞,以溶剂pH7.8的磷酸盐缓冲液为平行对照,制备作用后的细胞裂解液,提取mRNA并逆转录为cDNA,经RsaⅠ酶切后,将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性聚合酶链反应(PCR),将产物与T/A载体连接,构建cDNA消减文库,并
[Abstract]:Viral hepatitis and its related cirrhosis have a high incidence in our country, which seriously threaten the health of the people and bring a heavy economic burden to the patients, but there is no effective treatment yet. Whether chronic viral hepatitis or liver cirrhosis, there are various degrees of liver cell damage, in the comprehensive treatment of viral hepatitis, the promotion of hepatocyte regeneration plays an important role, especially for severe liver disease. The augmenter of liver regeneration (Augmenter if liver regeneration) plays an important role in the process of hepatocyte regeneration. ALR is a newly cloned protein factor, which can specifically stimulate the proliferation of hepatocytes. And in the animal model of acute liver failure and chronic liver disease treatment. However, the mechanism of action of ALR is not clear. The study of the mechanism of ALR will provide a theoretical basis and new ideas for the treatment of liver disease. Objective 1) to screen recombinant human aumenter of liver regeneration growth factor (rhALR) transactivated gene by suppression subtractive hybridization. To investigate the mechanism of rhALR stimulating hepatocyte regeneration. (2) to study the transactivation of cyclin dependent kinase 1 (cdk1) gene by recombinant human augmenter of liver regeneration (rhALR). Methods 1) Recombinant human liver regeneration enhancer protein was expressed and purified by Escherichia coli system. In vitro, HepG2 cells were stimulated with phosphate buffer of solvent pH7.8 as parallel control. The mRNA was extracted and reverse transcripted to cDNA. the experimental group cDNA was divided into two groups after being digested by Rsa 鈪，

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