The role of heterogeneity of cholangiocytes in immune-mediated cholangiopathy

目的：我们已经证明，主要分别位于肝内大胆管和小胆管的大胆管细胞和小胆管细胞具有不同的功能和对损伤的反应。然而，目前还没有系统的研究小和大胆管细胞之间的分子差异，这将解释胆管细胞的异质性。为了评估小胆管细胞和大胆管细胞之间的差异基因表达，进行了微阵列分析。方法：从正常小鼠（BALB/c）中分离小和大胆管细胞的原代培养物，并通过引入SV4O大T抗原基因使其永生化。克隆后，建立了小胆管细胞系和大胆管细胞系。通过电子显微镜（EM）和测量跨上皮电阻（TER）和促分泌素刺激的cAMP水平证实了它们的特征。分离的总RNA与微阵列（Atlas Glass Array Mouse 1.0和3.8）杂交，可检测4850个cDNA表达。杂交后，用GenePix荧光扫描仪扫描荧光信号，并用ArrayGauge软件分析。研究结果：EM，TER和分泌素刺激的cAMP合成是一致的概念，即小和大的永生化胆管细胞分别来自小和大的管道。当采用表达信号差异3.0倍的截止值时，4，850个cDNA中的230个cDNA（4.74%）在小胆管细胞和大胆管细胞之间差异表达。在这230个cDNA中，水通道蛋白8、IL-2受体B链和Caspase 9在大胆管细胞中表达更强。结论：微阵列成功地显示了小胆管细胞和大胆管细胞之间的特征性差异cDNA表达。这项技术提供了分子信息，进一步支持我们的假设，小和大的胆管有不同的功能。

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.