Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice.
OBJECTIVE
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 cholangiocyte 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 the introduction of the SV40 large T antigen gene. After cloning, small and large cholangiocyte 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 a 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 a cut-off value at the expression signal difference of 3.0 times was employed, 230 cDNAs among 4850 cDNAs (4.74%) were differentially expressed between small and large cholangiocytes. Of these 230 cDNAs, aquaporin 8, IL-2 receptor beta 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 bile ducts have different functions.
Division of Gastroenterology, Tohoku University School of Medicine, Seiryo, Aobaku, Sendai, Japan. yueno@mail.cc.tohoku.ac.jp
Yoshiyuki
Y
Ueno
Gianfranco
G
Alpini
Kaichiro
K
Yahagi
Noriatsu
N
Kanno
Yuki
Y
Moritoki
Koji
K
Fukushima
Shannon
S
Glaser
Gene
G
LeSage
Tooru
T
Shimosegawa
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D001653:Bile Ducts, Intrahepatic / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D019976:Cloning, Organism; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis; D012633:Secretin / Q000494:pharmacology; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D018076:DNA, Complementary; D012333:RNA, Messenger; D012633:Secretin; D000242:Cyclic AMP