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

为了探讨肝干细胞的分化机制，我们采用激光捕获显微解剖（LCM）、T7接头连接PCR扩增（TALPAT）和抑制减法杂交（SSH）技术对肝干细胞中差异表达基因进行了综合分析。这些细胞是我们建立的肝干细胞样细胞系的表型之一。我们用LCM法在该细胞系培养中选择性地提取目标干细胞样细胞，并提取其总RNA。由于其含量极低，经TALPAT扩增，获得了足够的量。接下来，我们在肝干细胞样细胞的TALPAT产物作为测试物和肝细胞的TALPAT产物作为驱动物之间进行SSH。一个测试程序和一个驱动程序- ssh产品分别作为一个正向减去cDNA和反向减去cDNA。用这些缺失的cDNA进行差异筛选，筛选出约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搜索确定相应的蛋白。此外，使用这两种TALPAT产品作为探针进行了虚拟反向北转，并选择了大约30个基因作为肝干细胞样细胞中的差异表达基因。其中4个基因在肝细胞中而非肝细胞中高表达，在13株肝癌细胞系非实质部分第一代聚集体中高表达，并有差异表达。这些结果提示这些基因可用于研究肝癌发生和分化的机制。

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.