| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Hepatic progenitor cells grew more rapidly than hematopoietic cells. After 10days culture, cells located in the center of aggregates for were stained for albumin, but peripheral cells for CK19. Adenovirus-mediated HNF-4 gene transfer resulted in increases in the expressions of HNF-4, ApoA1 ApoC3, PXR, TAT mRNA. Mice treated with HNF-4-transfected progenitor cells significantly survived than control mice (P=0.032). Plasma levels of albumin, total cholesterol, and glucose were higher in mice treated with cells transtected by HNF-4 than in control mice.
Using a cDNA microarray analysis, we identified that phosphatidylinositol 4,5-bisphosphate (PI (4,5)P_2) phosphatase mRNA was up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, which led to the finding that there was a decrease in the PI(4,5)P_2 levels of hepatocytes on EHS gel. These changes in hepatocytes on the EHS gel were accompanied by the promotion of the actin depolymerization and di
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fferentiated phenotypes of hepatocytes. Treatment with PI(4,5)P_2 or a phospholipase C inhibitor, U73122, resulted in a decrease in the mRNA expressions of albumin and hepatocyte nuclear factor 4 (HNF-4) in hepatocytes. In contrast, the actin-disrupting agent gelsolin increased the mRNA expressions of albumin and HNF-4. These findings indicated that the organization of the actin cytoskeleton via PI(4,5)P_2 is involved in the regulation of hepatocyte differentiation by the ECM.
To elucidate the relationship, we used small interfering RNA (siRNA), which obtains strong and specific knockdown of gene expression in cell culture. Treatment with siHNF-4 declined the expression levels for HNF-4mRNA in primary rat hepatocytes. The mRNA expression of albumin that was up-regulated in hepatocytes cultured on EHS gel, was decreased in the presence of siHNF-4. Moreover, siHNF-4 did not affect the morphology and actin assembly of hepatocytes. These findings demonstrated that HNF-4 directly regulates liver-specific gene expression and might be downstream of cytoskeletal organization in the mechanism by which the differentiated phenotype of hepatocytes is regulated by EHS gel. Less
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