【作者】 齐蕊；

【导师】 王勤；

【作者基本信息】 兰州大学 ， 生物物理学， 2006， 硕士

【摘要】 小鼠酒精性肝损伤分子生物学检测指标的探讨 目前酒精性肝损伤的发病率呈逐年上涨趋势,对酒精性肝病的诊断需要多种指标相互验证。本文拟探讨若干基因作为肝损伤分子生物学检测候选指标的可行性。首先取小鼠120只随机分成正常组和肝损伤模型组,连续给与模型组小鼠灌喂酒精。于第6周、第12周检测到模型组小鼠血清ALT活性有所升高,TP、ALB含量显著下降,而GLB含量无明显变化;肝组织病理切片显示肝细胞结构被破坏,有脂肪变性以及淋巴细胞浸润等特征。提示酒精诱导的小鼠肝损伤模型成立。然后分别提取模型组和正常组小鼠肝组织RNA,经SMART技术反转录成cDNA。在正常组和模型组cDNA中,用PCR扩增CD14、LBP、MCP-1、ICAM-1、IL-10、OPN 6个基因片断。结果显示以上基因在模型组中的表达均显著高于正常组;且随损伤程度加深,LBP、MCP-1、ICAM-1、IL-10表达量亦呈增加趋势。因此,这些基因可作为用分子生物学方法检测酒精性肝损伤的候选指标。本实验为在分子水平上进行肝损伤的检测提供理论依据。 用SSH方法构建CCl₄诱导小鼠急性肝损伤组织与正常肝组织差异表达基因的cDNA文库 本文利用抑制性消减杂交方法构建了CCl₄诱导小鼠急性肝损伤组织与正常肝组织差异表达基因的cDNA文库。首先建立CCl₄诱导急性肝损伤的小鼠模型:小鼠腹腔注射40%CCl₄:花生油溶液（40:60）,16h后检测到血清中AST、ALT活性均明显升高,TP、ALB含量显著下降,GLB含量无明显变化;病理切片显示肝细胞有气球样变性、小叶结构模糊、血管充血、部分淋巴细胞浸润。提示CCl₄诱导的急性肝损伤小鼠模型成立。然后我们在该模型中寻找与正常鼠肝组织差异表达的基因并构建消减cDNA文库:（1）分别提取肝组织mRNA,用SMART技术反转录成cDNA,经过酶切、接头连接、两轮消减杂交及两轮抑制性PCR,使得差异表达的cDNA片段得以富集;（2）用T/A克隆构建差异表达基因的cDNA消减文库;（3）随机挑选60个阳性克隆进行PCR鉴定,其中有55个克隆有200-750bp的插入片断,证实建库成功。该cDNA文库的构建为进一步筛选出代表急性肝损伤组织与正常肝组织差异表达的cDNA片段,并对这些cDNA进行序列测定、序列同源性分析以及进行完整基因的克隆、鉴定奠定了基础;为基因药物和诊断基因芯片的研发提供了依据。更多还原

【Abstract】 Study on several molecular indexes in diagnosis of alcoholic liver disease in miceThe mice model of alcoholic liver injury should be set up to evaluate several genes could be adapted for candidate indexes for diagnosing alcoholic liver disease. The mice were treated with alcohol. After 6 weeks and 12 weeks, compared with the normal mice, the activity of alanine aminotransferase （ALT） in serum in alcohol-treated mice was increased;while the concentrations of total proteins （TP）, albumin（ALB） were reduced markedly. In pathological sections of liver tissue, destruction of the structure of hepatocytes, steatosis, and lymphocytes soakage were found in mice treated with alcohol. The mice model of alcoholic liver injury was established successfully. Then, RNA was extracted from liver tissues of normal mice and alcohol-treated mice, and was reverse-transcripted into cDNA. Six genes such as CD14, LBP, MCP-1, ICAM-1, IL-10, OPN were amplified by PCR in liver tissues of normal mice and alcohol-treated mice respectively. These genes of alcohol-treated mice were expressed at levels higher than those of normal mice. Thus, the results suggest these genes could be the candidate indexes in diagnosis of alcoholic liver disease by molecular biology method.Construction of the subtracted cDNA library of differentially expressed genes in mice liver of CCl₄-induced acute injury and normal liverHuman genomic technology make the differential expression of function genes get more and more popular. To find differentially expressed genes becomes most hot in the molecular biology research. Here, we make the differentially expressed cDNA subtracted library in mice liver treated with CCl₄ compared with normal mice liver. First, mice model of CCl₄-induced acute liver injury should be set up. Mice were intraperitoneally （ip.） administered 40% CCl₄: peanut oil. After sixteen hours, compared with normal mice, the activities of aspartate aminotransferase （AST）, alanine aminotransferase （ALT） in serum were increased in model mice, while the concentrations of total proteins（TP）, albumin（ALB） in serum were reduced markedly. In pathological sections, the blur of hepatic lobule, the hyperemia, the ballooning degeneration of hepatocyte were found in the liver tissues of the mice treated with CCl₄. The mice model of CCl₄-induced acute liver injury was set up successfully. To find the differentially expressed genes in mice liver treated with CCl₄ compared with normal mice liver and make the differentially expressed cDNA subtracted library, suppression subtractive hybridization （SSH） was applied. The differentially expressed fragments were obtained through twice hybridizations and twice PCR, then T/A cloning was accomplished to construct the cDNA library. Last, sixty positive cloneswere randomly picked out and identified by PCR, Fifty-five contain positive insert fragments which range from 200 to 750 bp. A subtracted cDNA library of differentially expressed genes in mice liver of CCU-induced acute injury and normal liver is successfully constructed with SSH and T/A cloning techniques. This experiment has prepared for the cloning and identification of the full-length genes which were differentially expressed. Furthermore, it also helps to exploit gene remedy and diagnosis gene chip.更多还原