脂肪酸诱导鹅肝细胞差异表达基因的筛选和鉴定
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摘要
在动物肝细胞内,甘油三酯(TG)的异常沉积导致肝脂肪变性。将游离脂肪酸(FFAs)添加到肝细胞中可导致肝脂肪变性,但是关于FFAs与TG的异常沉积之间联系和脂肪沉积相关基因的差异表达的机制目前还不清楚。本研究以四川白鹅(Ansercygnoides)为研究对象,利用抑制消减杂交技术构建了FFAs诱导的肝脂肪变性细胞和正常肝细胞差异cDNA文库,对文库中序列进行了功能注释和分类,初步揭示了FFAs诱导后肝细胞差异表达基因的种类和数量,这为从分子水平探讨肥肝的机理奠定了基础。本研究取得的主要结果如下:
1.采用添加1mM游离脂肪酸混合物FFAs到鹅原代肝细胞中培养36h,成功建立肝细胞脂肪变性的细胞模型。
2.应用抑制性消减杂交技术成功构建了正常肝细胞与脂肪变性肝细胞正反向SSH文库。正向文库(SSH-1)扩增后获得1344个克隆,反向文库(SSH-2)扩增后获得1152个克隆。应用斑点杂交筛选阳性克隆测序,SSH-1文库有30个测序成功,SSH-2文库有23个测序成功。
3.对去除载体序列和接头序列后的EST通过BLASTn进行在线DNA序列同源性比较分析得到:SSH-1获得23个已知基因,占克隆测序的76.7%;已知EST有6个占20%;全新EST有1个占3.3%。其中SSH-2文库已知基因有18个占克隆测序的78.3%;已知EST有1个占4.3%,全新EST有4个占17.4%。
4.SSH文库共获得已知基因41个,对EST功能分类发现,细胞结构和运动占已知基因的2%,细胞信号和传导占12%,翻译核糖体结构和功能占25%,细胞转录占5%,细胞凋亡占2%,物质转运占17%,物质代谢占22%,未知基因占15%。
5.ALDOB、TGM3、RPL家族(RPL22、RPL23、RPL39、RPL5)、APoH等这些差异表达的EST序列与新基因片断在肝细胞的生长或脂肪代谢等过程中起了重要作用,为进一步研究肝脂肪变性提供了线索。
Hepatic steatosis results from the excessive accumulation of triglyerides(TG) in the animal liver.FFAs cultured with Hepatocytes could induce Hepatoeytes steatosis, but it has been not clear about the relation between FFAs and TG abnormal deposition,and the mechanism of differentialy expressed genes in fat deposition.In this study,using the Sichuan White geese as material,we construct two forward and reverse SSH cDNA libraries between the normal hepatocytes and the steatosis hepatocytes.By homology alignment and function annotation,we discoverd the sorts and quantity of differentialy expressed genes in FFAs induced hepatocytes,which would provide a bias for elucidating the mechanism of fatty liver in the molecular level.The Results were shown as follows:
1.The hepatocyte steatosis models were established successfully induced by 1mmol/ml FFAs for 36d.
2.Differentially expressed cDNA library were constructed using suppression subtractive hybridization.1344 positive clones were obtained in SSH-1,1152 positive clones were obtained inSSH-2.SSH-1 library had 30 successful sequences,and SSH-2 library has 23 successful sequences.
3.A search for sequence homology in the GenBank and EST database by BLASTn revealed that the distribution of SSH-1 EST clusters for high homology genes.The high homology genes,high homology ESTs and novel ESTs were23,6 and 1,and the proportion were 76.7%,20%and 3.3%.In SSH-2,the high homology genes and novel ESTs were 19,land 4,and the proportion were 78.3%,4.3%and 17.4%.
4.the proportions of the high homology genes was 41 in SSH library.Categories of functions ESTs:the proportions of cell structure/movement was 2%,the cell signal/conduction was 12%,slation/expression regulation was 25%,transporters was 5%cell apoptosis was 2%, material conveying was17%,metabolismd and unknow gene were 22%and15%.Of course, the genes involved in metabolism and transporters were rich expressed.
5.Roles of different character feature of partial diferentially expreesed genes in two geese breeds were presumed based on references and our research results.RP (RPL22,RPL23,RPL39,RPL5),TGM3,FABP,THBS1,and APOH were considered playing an important role in different character feature in different geese.They are the biological function of providing the material and they provide a clue in searching the important genes related to the hepatic steatosis and the foie gras.
1.采用添加1mM游离脂肪酸混合物FFAs到鹅原代肝细胞中培养36h,成功建立肝细胞脂肪变性的细胞模型。
2.应用抑制性消减杂交技术成功构建了正常肝细胞与脂肪变性肝细胞正反向SSH文库。正向文库(SSH-1)扩增后获得1344个克隆,反向文库(SSH-2)扩增后获得1152个克隆。应用斑点杂交筛选阳性克隆测序,SSH-1文库有30个测序成功,SSH-2文库有23个测序成功。
3.对去除载体序列和接头序列后的EST通过BLASTn进行在线DNA序列同源性比较分析得到:SSH-1获得23个已知基因,占克隆测序的76.7%;已知EST有6个占20%;全新EST有1个占3.3%。其中SSH-2文库已知基因有18个占克隆测序的78.3%;已知EST有1个占4.3%,全新EST有4个占17.4%。
4.SSH文库共获得已知基因41个,对EST功能分类发现,细胞结构和运动占已知基因的2%,细胞信号和传导占12%,翻译核糖体结构和功能占25%,细胞转录占5%,细胞凋亡占2%,物质转运占17%,物质代谢占22%,未知基因占15%。
5.ALDOB、TGM3、RPL家族(RPL22、RPL23、RPL39、RPL5)、APoH等这些差异表达的EST序列与新基因片断在肝细胞的生长或脂肪代谢等过程中起了重要作用,为进一步研究肝脂肪变性提供了线索。
Hepatic steatosis results from the excessive accumulation of triglyerides(TG) in the animal liver.FFAs cultured with Hepatocytes could induce Hepatoeytes steatosis, but it has been not clear about the relation between FFAs and TG abnormal deposition,and the mechanism of differentialy expressed genes in fat deposition.In this study,using the Sichuan White geese as material,we construct two forward and reverse SSH cDNA libraries between the normal hepatocytes and the steatosis hepatocytes.By homology alignment and function annotation,we discoverd the sorts and quantity of differentialy expressed genes in FFAs induced hepatocytes,which would provide a bias for elucidating the mechanism of fatty liver in the molecular level.The Results were shown as follows:
1.The hepatocyte steatosis models were established successfully induced by 1mmol/ml FFAs for 36d.
2.Differentially expressed cDNA library were constructed using suppression subtractive hybridization.1344 positive clones were obtained in SSH-1,1152 positive clones were obtained inSSH-2.SSH-1 library had 30 successful sequences,and SSH-2 library has 23 successful sequences.
3.A search for sequence homology in the GenBank and EST database by BLASTn revealed that the distribution of SSH-1 EST clusters for high homology genes.The high homology genes,high homology ESTs and novel ESTs were23,6 and 1,and the proportion were 76.7%,20%and 3.3%.In SSH-2,the high homology genes and novel ESTs were 19,land 4,and the proportion were 78.3%,4.3%and 17.4%.
4.the proportions of the high homology genes was 41 in SSH library.Categories of functions ESTs:the proportions of cell structure/movement was 2%,the cell signal/conduction was 12%,slation/expression regulation was 25%,transporters was 5%cell apoptosis was 2%, material conveying was17%,metabolismd and unknow gene were 22%and15%.Of course, the genes involved in metabolism and transporters were rich expressed.
5.Roles of different character feature of partial diferentially expreesed genes in two geese breeds were presumed based on references and our research results.RP (RPL22,RPL23,RPL39,RPL5),TGM3,FABP,THBS1,and APOH were considered playing an important role in different character feature in different geese.They are the biological function of providing the material and they provide a clue in searching the important genes related to the hepatic steatosis and the foie gras.
引文
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