权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of differentially expressed genes in hepatic stem-like cells by cDNA subtraction.

通过cDNA消减法分析肝干样细胞中差异表达基因。

基本信息

批准号：
14570178
负责人：
KANO Junko
金额：
$ 2.3万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570178/
关键词：
hepatic stem cell differentially expressed gene cDNA subtraction laser capture microdissection Laser capture microdissection 肝臓幹細胞分化

项目摘要

To investigate the differentiation mechanism of hepatic stem cells, we examined the comprehensive analysis of differentially expressed genes in them by laser capture microdissection (LCM), T7 adaptor ligation PCR amplification T7 (TALPAT) and suppression subtractive hybridization (SSH). These cells were one of the phenotypes constituting a hepatic stem-like cell line that we established. We picked up selectively target stem-like cells in the culture of this cell line by LCM, and extracted their total RNA. Because of its very small amount, it was amplified by TALPAT, and sufficient amount were obtained. Next, we performed SSH between TALPAT product of hepatic stem-like cells as a tester and TALPAT product of hepatocytes as a driver. A tester-and a driver-SSH product were acquired as a forward subtracted cDNA and a reverse subtracted cDNA, respectively. Differential screening with these subtracted cDNA was performed, and about 40 clones were selected as target ones. Sequences of these clones were analyzed, and the cording proteins were decided by Blast search. Furthermore, virtual reverse northern used both TALPAT products as probes were performed, and about 30 genes were selected as differentially expressed genes in hepatic stem-like cells. Among them, 4 genes which expressed highly in them rather than in hepatocytes were selected, and were demonstrated high expression in the aggregates of the first passage of nonparenchymal fraction, and differential expression in 13 hepatocellular carcinoma lines. These results suggested the genes were utilized to the studies for the mechanisms of hepatocarcinogenesis and differentiation.

为了研究肝干细胞的分化机制，我们通过激光捕获显微切割（LCM）、T7接头连接PCR扩增T7（TALPAT）和抑制消减杂交（SSH）对肝干细胞中的差异表达基因进行综合分析。这些细胞是构成我们建立的肝干样细胞系的表型之一。我们通过LCM选择性地在该细胞系的培养物中挑选出目标干细胞，并提取它们的总RNA。由于其量非常少，通过TALPAT进行扩增，得到了足够的量。接下来，我们在作为测试器的肝干细胞样细胞的TALPAT产品和作为驱动器的肝细胞的TALPAT产品之间进行SSH。分别以正向消减 cDNA 和反向消减 cDNA 的形式获得测试器-SSH 产物和驱动器-SSH 产物。用这些消减的cDNA进行差异筛选，选择约40个克隆作为目标克隆。对这些克隆进行序列分析，并通过Blast搜索确定编码蛋白。此外，Virtual Reverse Northern使用两种TALPAT产品进行探针检测，筛选出约30个基因作为肝干样细胞中的差异表达基因。其中，选择了4个在细胞内而不是在肝细胞中高表达的基因，并在第一代非实质部分的聚集体中表现出高表达，并在13个肝细胞癌细胞系中表现出差异表达。这些结果表明这些基因可用于肝癌发生和分化机制的研究。