Analysis of differentially expressed genes in hepatic stem-like cells by cDNA subtraction.
| Project/Area Number |
14570178
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Experimental pathology
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| Research Institution | University of Tsukuba |
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Principal Investigator |
KANO Junko University of Tsukuba, Graduate School of Comprehensive Human Science, Assistant Professor, 基礎医学系, 講師 (60334059)
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| Co-Investigator(Kenkyū-buntansha) |
NOGUCHI Masayuki University of Tsukuba, Graduate School of Comprehensive Human Science, Professor, 基礎医学系, 教授 (00198582)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | hepatic stem cell / differentially expressed gene / cDNA subtraction / laser capture microdissection / Laser capture microdissection / 肝臓 / 幹細胞 / 分化 |
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| Research Abstract |
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.
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Report
(3 results)
Research Products
(8 results)
