【作者】 杨玉琴；

【导师】 高诚； 谢建云；

【作者基本信息】 复旦大学 ， 动物学， 2010， 硕士

【摘要】 目的：非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)发病率逐年增高,且有进展到肝纤维化肝硬化的趋势,目前其确切的发病机制尚不清楚。我们在以往的实验中发现东方田鼠有易发脂肪肝的倾向,因此建立东方田鼠非酒精性脂肪肝模型,利用SMART (Switching Mechanism at 5’end of RNA Transcript, RNA5’端转录机制)技术构建了东方田鼠肝脏cDNA (complementary DNA,互补DNA)质粒文库,在此基础上筛选与东方田鼠脂肪肝发病相关的基因,并获得其全长序列,为进一步研究非酒精脂肪肝发病机制奠定基础。方法：选取105只6周龄洞庭湖种群雄性东方田鼠,随机分成三组,每组35只,分别为对照组、模型组1和模型组2,各组分别饲喂高纤维料(在大小鼠基础料的基础上加粗纤维,使粗纤维含量为10%,粗脂肪含量为3%)、高脂料(在大小鼠基础料的基础上加5%猪油、0.5%胆固醇和0.1%胆盐)、大小鼠基础料。各组分别于造模开始后第2、4、6、8、12周处死,称量体重及肝重,计算肝指数,采血检测东方田鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、甘油三酯(TG)、总胆固醇(TC)、碱性磷酸酶(ALP)、游离脂肪酸(FFA)、血糖(GLU)、r-谷氨酰转移酶(r-GGT)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)和胆碱酯酶(CHE),并取肝脏HE染色后做病理学观察。通过文献确定,可能与非酒精性脂肪肝相关的基因,参照NCBI中大小鼠基因cDNA序列的保守区设计其特异性引物,进行实时荧光定量PCR检测,比较基因动态表达水平。选取12月龄洞庭湖种群东方田鼠4只,取肝脏组织,提取总RNA,用SMART技术构建东方田鼠肝脏cDNA质粒文库,测定文库滴度和容量,并随机挑选单个菌落测序,计算全长基因的比率根据实时荧光定量PCR结果确定目的基因,通过PCR方法筛选cDNA质粒文库,获得目的菌落,用pBluescriptⅡSK通用引物M13R测序,获得基因的全长序列,并进行序列分析比较。结果：模型组1东方田鼠肝细胞均呈现弥漫性脂肪变性,模型组2东方田鼠肝细胞脂肪变性较轻,对照组东方田鼠肝脏基本正常。与对照组相比,模型组1肝重和肝指数都明显升高(P<0.05),血清ALT、AST、TC、FFA、r-GGT、LDL、CHE和GLU都明显升高(P<0.05),HDL和TG均明显降低(P<0.05),ALP无明显差异(P>0.05)；对8个基因进行了实时荧光定量PCR结果显示肝组织中的CYP2E1(细胞色素P4502E1)、ECHS1(烯酰辅酶A水合酶短链1)、CYP2D5(细胞色素P4502D5)、CYP2a3a(细胞色素P4502a3a)和PGC-1(过氧化物酶体增殖物激活受体辅激活因子1)这5基因表达上调(P<0.05),SC4MOL(固醇甲基氧化酶)、Adipor(脂联素受体)和AMPK-1(腺苷酸活化激酶-1)这3个基因表达下调(P<0.05)。与对照组相比,模型组2肝重和肝指数明显升高(P<0.05),血清FFA、LDL、CHE显著升高(P<0.05),HDL明显降低(P<0.05),其余指标与对照组相比无显著差异；实时荧光定量PCR结果显示所测基因未有明显改变(P>0.05)。利用SMART技术构建了东方田鼠肝脏cDNA质粒文库,该cDNA文库滴度为1076 pfu/μL,重组率约94%,库容量达1.08×106。测定了22个克隆序列经BLAST分析,与GenBank中大鼠、小鼠、田鼠以及人类已知功能蛋白具有75%以上的同源性,基因完整性比率为77.3%。克隆了CYP2E1、CYP2D5、ECHS1三个基因,并获得了它们的全长cDNA序列。CYP2E1基因cDNA全长为1685bp,有1482bp的完整开放阅读框,编码494个氨基酸：CYP2D5基因cDNA全长为1690bp,有1514bp的完整开放阅读框,编码504个氨基酸；ECHS1基因cDNA全长为1013bp,有873bp的完整开放阅读框,编码290个氨基酸。这三个基因的核酸序列及其推测的氨基酸序列与人、大小鼠序列高度同源。在GenBank中注册号分别为GQ507485、GQ507486、GQ845171。更多还原

【Abstract】 Objective:The incidence of Non-alcoholic fatty liver (nonalcoholic fatty liver disease, NAFLD) increased year by year, and the disease can progress to liver fibrosis and cirrhosis, but its precise mechanism is currently unclear. Our early experiment showed that Microtus fortis is prone to fatty liver, therefore, the purpose of this study is to establish a Non-alcoholic fatty liver model of Microtus fortis, and to study the pathogenesis of Non-alcoholic fatty liver disease in the model of Microtus fortis.Methods:We selected 105 male Microtus fortis in Dongting Lake which are 6-week-old, and randomly divided them into three groups.The three groups were fed the high-fat ingredients (plus 5% lard,0.5% cholesterol and 0.1% bile salt into based on the standard diet for rats and mice, model 1 group), the standard diet (model 2 group), the high-fiber material (the final concentration of Bold fiber was 10%, crude fat was 3% on the basis of the standard diet for rats and mice, control group) respectively.7 voles of each group were sacrificed at the beginning of the study time of 2,4,6,8,12week. During the process of our study we had observed the general situation and changes in liver index of Microtus fortis. ALT, AST, TG, TC, ALP, FFA, r-GT, HDL, LDL, CHE, GLU values were measured by biochemistry analyzer. The histological changes of all voles’livers were observed using light microscopy. Genes which should be possible related to non-alcoholic fatty liver were selectecd. The cDNA-specific primers were designed in the conserved region according to the sequences of mice and rats from the NCBI. Then real-time PCR was done to obtain the gene expression levels of the livers.We selected four Microtus fortis in Dongting Lake which are 12 months of age, after we sacrificed them whichever liver were obtained to extracte total RNA, then The plasmid cDNA library from liver of Microtus fortis was constructed by using SMART technology. The titers and capacity of this cDNA library were determined. Randomly selected a single colony to sequence and calculate the ratio of full-length gene.The plasmid cDNA library of the liver from Microtus fortis was constructed by using SMART technology. The purposed colonies were got through screening libraries by PCR method, and their full-length cDNA sequences were obtained by sequencing with pBluescriptⅡSK universal primers M13R.Results:Voles fed the high-fat diet developed panlobular macrovesicular steatosis, voles fed the standard diet developed light steatosis, whereas those fed the high-fiber diet had normal livers.Compared with the control group, liver weight and liver index of model group 1 were significantly increased (P< 0.05), serum ALT, AST, TC, FFA, r-GT, LDL, CHE and GLU were significantly high (P< 0.05), HDL and TG were significantly low (P < 0.05). Real-time PCR showed that the five genes of CYP2E1, ECHS1, CYP2D5, CYP2a3a and PGC-1 were high-expression (P< 0.05), the three genes of SC4MOL, Adipor and AMPK-1 were low-expression (P< 0.05).Compared with control group, the liver weight and liver index of model group 2 were significantly increased (P< 0.05), the FFA, LDL and CHE also significantly increased (P< 0.05), HDL of blood decreased significantly (P< 0.05), other indexes had no significant difference. Real-time PCR showed that all the gene expression trends are not obvious (P> 0.05).cDNA plasmid library of Microtus fortis liver was constructed by using SMART technology. The titers of this cDNA library were 1076 pfu/ul, the recombination rate was about 94%, the library capacity was 1.08×106, the integrity rate was 77.3%.Three full-length cDNA sequences of Microtus fortis CYP2D5, CYP2E1 and ECHS1 were obtained. The CYP2D5 cDNA is 1865bp in length and contains a 1482bp open reading frame (ORF) encoding a 494 amino acids. The CYP2D5 cDNA is 1690bp in length, and contains a 1514bp ORF encoding 504 amino acids. The ECHS1 cDNA is 1013bp in length, and contains an 873bp ORF encoding 290 amino acids. Sequence analysis reveals that the identity of the three cDNA sequences and deduced amino acids among Microtus fortis, Homo saplens, Mus musculus and Rattus norvegicus is high.We have registerd the three full-length cDNA sequences in the GenBank, registration are respectively GQ507485, GQ507486, GQ845171.更多还原