本文選題：重組人肝再生增強(qiáng)因子 + 細(xì)胞周期蛋白依賴性激酶1��； 參考：《鄭州大學(xué)》2005年碩士論文

【摘要】：病毒性肝炎及其相關(guān)的肝硬化,在我國發(fā)病率高、危害嚴(yán)重,嚴(yán)重威脅著人民群眾的身體健康,并給病人帶來了沉重的經(jīng)濟(jì)負(fù)擔(dān),但目前還沒有確切有效的治療手段。無論是慢性病毒性肝炎,還是肝硬化,都存在不同程度的肝細(xì)胞損傷,在病毒性肝炎的綜合治療方案中,促進(jìn)肝細(xì)胞再生占有重要的地位,特別是對于重型肝病。肝再生增強(qiáng)因子(Augmenter if liver regeneration,ALR)在肝細(xì)胞的再生過程中發(fā)揮著舉足輕重的作用。ALR是新近克隆的蛋白質(zhì)因子,能特異地刺激肝源細(xì)胞的增殖,并在動物模型中對急性肝衰竭和慢性肝病有救治作用。但其作用機(jī)制尚不清楚,開展ALR作用機(jī)制的研究,將為肝病的治療提供理論依據(jù)和新思路。 目的 (1) 應(yīng)用抑制性削減雜交技術(shù)篩選重組人肝再生增強(qiáng)因子(recombinant human aumenter of liver regeneration,rhALR)反式激活的基因,探討rhALR刺激肝細(xì)胞再生的作用機(jī)制;(2) 研究重組人肝再生增強(qiáng)因子(rhALR)對細(xì)胞周期蛋白依賴性激酶1(cdk1)基因的反式激活作用。 方法 (1) 應(yīng)用大腸桿菌系統(tǒng),表達(dá)、純化重組人肝再生增強(qiáng)因子蛋白。體外刺激HepG2細(xì)胞,以溶劑pH7.8的磷酸鹽緩沖液為平行對照,制備作用后的細(xì)胞裂解液,提取mRNA并逆轉(zhuǎn)錄為cDNA,經(jīng)RsaⅠ酶切后,將實驗組cDNA分成兩組,分別與兩種不同的接頭銜接,再與對照組cDNA進(jìn)行兩次消減雜交及兩次抑制性聚合酶鏈反應(yīng)(PCR),將產(chǎn)物與T/A載體連接,構(gòu)建cDNA消減文庫,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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