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

点击翻译按钮获取中文摘要

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.