The role of heterogeneity of cholangiocytes in immune-mediated cholangiopathy

目的：我们已经证明，主要位于大、小肝内胆管的大小胆管细胞具有不同的功能和对损伤的反应。然而，目前还没有关于大小胆管细胞之间分子差异的系统研究，这可以解释胆管细胞的异质性。为了评估大小胆管细胞之间的差异基因表达，进行了基因芯片分析。方法：从正常小鼠(BALB/c)分离原代培养的大小胆管细胞，并导入SV4O大T抗原基因使其永生化。克隆后，建立了大、小胆管细胞系。其特征通过电子显微镜(EM)、跨皮细胞电阻(TER)和促胰液素刺激的cAMP水平的测定得到证实。将分离的总RNA与基因芯片(Atlas Glass Array Mouse 1.0和3.8)杂交，检测4850个基因的表达。杂交后，用GenePix荧光扫描仪扫描荧光信号，用ArrayGauge软件进行分析。结果：EM、TER和促分泌素刺激的cAMP合成分别与大、小永生化胆管细胞起源于大、小胆管的观点一致。以表达信号差3.0倍为界值时，4850个cDNA中有230个(4.74%)在大小胆管细胞中有差异表达。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较强。结论：基因芯片成功地显示了大小胆管细胞之间的差异表达。这项技术提供了分子信息，进一步支持了我们的假设，即小胆管和大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.