|Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
AIMS : We have shown that large and small cholangiocytes which reside primarily in large and small intrahepatic bile ducts respectively have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocytes heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed. METHODS : Primary cultures of small and large cholangiocytes were isolated from normal mice (BALB/c), and immortalized by introduction of the SV4O large T antigen gene. After cloning, small and large cholangiocytes cell lines were established. Their characteristic features were confirmed by electron microscopy (EM) and measurement of transepithelial electrical resistance (TER), and secretin-stimulated cAMP levels. Isolated total RNAs were hybridized with microarrays (Atlas Glass Array Mouse 1.0 and 3.8), which detects 4850 cDNA expressions. After hybridization, the fluorescent signals were scanned by GenePix fluorescent scanner and analyzed using ArrayGauge software. RESULTS : EM, TER and secretin-stimulated cAMP synthesis are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively. When cut-off value at the expression signal difference of 3.0 times was employed, 230 cDNAs among 4,850 cDNAs (4.74%) were differentially expressed between small and large cholangiocytes. Of these 230 cDNAs, aquaporin 8, IL-2 receptor b chain, and Caspase 9 were more strongly expressed by large cholangiocytes. CONCLUSIONS : Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes. This technique provides molecular information which further supports our hypothesis that small and large bileducts have different functions.