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

[背景与目的]慢性胰腺炎（chronic pancreatitis，CP）是一种进行性胰腺炎性疾病。囊性纤维化跨膜传导调节因子（CFTR）是一种在人胰管细胞顶端质膜中表达的环AMP调节的氯离子通道。最近的证据表明CFTR突变是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）提取患者白细胞基因组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)从豚鼠小肠中提取基因组DNA。采用PCR方法扩增豚鼠CFTR基因片段，并直接测序。基于人、大鼠和小鼠CFTR之间的共有序列设计PCR引物组。从豚鼠胰腺分离小叶间导管并培养过夜。采用RNA干扰技术降低豚鼠小叶间导管CFTR的表达。[结果]（1）在日本酒精性慢性胰腺炎患者中，发现CFTR基因的新突变（L1156 F）。L1156 F CFTR显示爪蟾卵母细胞中HCO 3-渗透性降低。(2)豚鼠CFTR由1481个氨基酸残基组成。在具有CFTR特异性RNAi的离体豚鼠胰管中的液体分泌速率降低至具有乱序RNAi的对照导管中的速率的一半。[结论]这些证据表明，由于突变或多态性导致CFTR表达减少导致胰腺导管功能障碍，并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.