Roles of ion channels and water channels in the development of chronic pancreatitis

【背景与目的】慢性胰腺炎是胰腺的一种进行性炎性疾病。囊性纤维化跨膜传导调节因子（CFTR）是一种环amp调控的氯离子通道，表达于人胰管细胞的顶质膜。最近的证据表明，CFTR突变是发生慢性胰腺炎的危险因素之一。本项目的目的是了解离子和水通道在慢性胰腺炎发展中的作用。【方法】(1)从患者白细胞中提取基因组DNA。通过直接测序鉴定CFTR突变。每个外显子片段通过聚合酶链反应扩增。(2)从豚鼠小肠中分离出基因DNA。用PCR扩增豚鼠CFTR片段并直接测序。根据人、大鼠和小鼠CFTR的一致序列设计PCR引物。从豚鼠胰腺中分离小叶间管，培养过夜。采用RNA干扰法降低CFTR在豚鼠小叶间管中的表达。【结果】(1)在日本酒精性慢性胰腺炎患者中发现CFTR （L1156F）新突变。L1156F CFTR显示爪蟾卵母细胞HCO3-通透性降低。(2)豚鼠CFTR由1481个氨基酸残基组成。转染CFTR特异性RNAi后，分离豚鼠胰管的液体分泌率降至转染紊乱RNAi的对照胰管的一半。[结论]这些证据提示CFTR基因突变或多态性导致的表达减少导致胰腺导管功能障碍，可能是日本人慢性胰腺炎的原因。

[Background and aim]Chronic pancreatitis is a progressive inflammatory disease of the pancreas. Cystic Fibrosis Transmembrane conductance Regulator(CFTR)is a cyclic AMP-regulated chloride channel expressed in the apical plasma membrane of human pancreatic duct cells. Recent evidence suggest that mutation(s) in CFTR is one of the risk factors in developing chronic pancreatitis. The aim of this project was to educidate the roles of ion and water channels in the development of chronic pancreatitis.[Methods] (1) Genomic DNA was extracted from the leukocytes of the patients. CFTR mutations was identified by direct sequencing. Each exon fragments were amplified by polymerase chain reaction. (2) Genonic DNA of Guinea-pig was isolated from the small intestine. Fragments of guinea pig CFTR was amplified by PCR and directly sequenced. PCR primer sets were designed based on the consensus sequence among human, rat and mouse CFTR. Interlobular ducts were isoloated from the Guinea pig pancreas and cultured overnight. RNA interference was used to reduce the expression of CFTR in guinea pig interlobular ducts.[Results] (1) In Japanese patient with alcoholic chronic pancreatitis, new mutation in CFTR (L1156F) was identified. L1156F CFTR showed the reduced HCO3- permeability in Xenopus oocytes. (2) Guinea pig CFTR consists of 1481 amino acid residues. Fluid secretary rate in isolated guinea pig pancreatic ducts with CFTR specific RNAi was decreased to half of the rate in control ducts with scrambled RNAi.[Conclusion] These evidence sugget that reduced expression of CFTR due to the mutations or polymorphisms causes the pancreatic ductal dysfunction and might be the cause of chronic pancreatitis in Japanese.