Zhu Hu, Zhu Jin Xia, Lo Pui Shan, Li Jianmin, Leung Ka Man, Rowlands Dewi Kenneth, Tsang Lai Ling, Yu Mei Kuen, Jiang Jian Li, Lam Sun Yee, Chung Yiu Wa, Zhou Zuomin, Sha Jiahao, Chang Chan Hsiao
Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, SAR, Shatin, Hong Kong, China.
Lancet. 2003 Dec 20;362(9401):2059-65. doi: 10.1016/s0140-6736(03)15100-8.
Cystic fibrosis is caused by mutations in the gene encoding an ion-transport protein, the cystic-fibrosis transmembrane conductance regulator (CFTR). Defective secretion of anions is the primary cause of many of the clinical manifestations of cystic fibrosis, including pancreatic insufficiency. We aimed to identify a molecular mechanism from which a new method to circumvent defective pancreatic secretion could be derived.
Multiple-human-tissue RT-PCR and semiquantitative RT-PCR analyses were used to examine gene expression. An antisense technique was used in conjunction with radioimmunoassay, Fura-2 spectrofluorometry, immunohistochemistry, and the short-circuit current technique (Ussing chamber) for elucidation of gene function and its application in rescuing defective pancreatic secretion.
We cloned a newly identified gene, NYD-SP27, which has structural similarity to an isoform of phospholipase C. NYD-SP27 was expressed endogenously in human pancreatic-duct cells and upregulated in cystic fibrosis. Suppression of NYD-SP27, by transfection of its antisense into human cystic-fibrosis pancreatic-duct cells, resulted in augmentation of phospholipase-C-coupled calcium-ion release and protein kinase C activity, improvement in the amount of mutated CFTR reaching the plasma membrane, and restoration of cAMP-activated pancreatic anion secretion.
NYD-SP27 exerts an inhibitory effect on phospholipase-C-coupled processes that depend on calcium ions and protein kinase C, including CFTR trafficking and function. Its upregulation in pancreatic-duct cells may reveal a previously unsuspected defect in cystic fibrosis contributing to pancreatic insufficiency, and thus represents a new target for pharmacological intervention in cystic fibrosis.
囊性纤维化由编码离子转运蛋白——囊性纤维化跨膜传导调节因子（CFTR）的基因突变引起。阴离子分泌缺陷是囊性纤维化许多临床表现的主要原因，包括胰腺功能不全。我们旨在确定一种分子机制，从中衍生出一种规避胰腺分泌缺陷的新方法。
采用多人组织逆转录聚合酶链反应（RT-PCR）和半定量RT-PCR分析来检测基因表达。运用反义技术结合放射免疫测定、Fura-2荧光分光光度法、免疫组织化学和短路电流技术（尤斯灌流小室）来阐明基因功能及其在挽救胰腺分泌缺陷中的应用。
我们克隆了一个新发现的基因NYD-SP27，它与磷脂酶C的一种同工型具有结构相似性。NYD-SP27在人胰腺导管细胞中内源性表达，在囊性纤维化中上调。将其反义寡核苷酸转染到人囊性纤维化胰腺导管细胞中抑制NYD-SP27，可导致磷脂酶C偶联的钙离子释放和蛋白激酶C活性增强，到达质膜的突变型CFTR数量增加，以及环磷酸腺苷（cAMP）激活的胰腺阴离子分泌恢复。
NYD-SP27对依赖钙离子和蛋白激酶C的磷脂酶C偶联过程发挥抑制作用，包括CFTR的转运和功能。它在胰腺导管细胞中的上调可能揭示了囊性纤维化中一个以前未被怀疑的导致胰腺功能不全的缺陷，因此代表了囊性纤维化药物干预的一个新靶点。
