The role of heterogeneity of cholangiocytes in immune-mediated cholangiopathy

目的：我们已经表明，主要存在于肝内大胆管和小胆管的大胆管细胞和小胆管细胞对损伤有不同的功能和反应。然而，目前还没有关于大、小胆管细胞分子差异的系统研究，这可以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）中分离大小胆管细胞原代培养，引入sv40o大T抗原基因永生化。克隆后，建立了大小胆管细胞细胞系。通过电镜（EM）、经上皮电阻（TER）测量和分泌素刺激的cAMP水平证实了它们的特征。将分离的总rna与微阵列（Atlas Glass Array Mouse 1.0和3.8）杂交，检测4850个cDNA表达。杂交后荧光信号用GenePix荧光扫描仪扫描，用ArrayGauge软件分析。结果：EM、TER和分泌素刺激的cAMP合成符合小、大永生化胆管细胞分别来源于小、大胆管的概念。采用表达信号差异3.0倍的临界值，4850个cdna中有230个（4.74%）在大、小胆管细胞间存在差异表达。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为强烈。结论：微阵列成功显示了大、小胆管细胞间cDNA表达差异的特征。这项技术提供的分子信息进一步支持了我们的假设，即小型和大型胆管具有不同的功能。

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.