siRNA 沉默 CFTR 基因或 CFTR 激活剂腔内应用对豚鼠胰腺导管细胞液体分泌的影响。

Effects of CFTR gene silencing by siRNA or the luminal application of a CFTR activator on fluid secretion from guinea-pig pancreatic duct cells.

机构信息

Department of Gastroenterology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.

出版信息

Biochem Biophys Res Commun. 2011 Jul 15;410(4):904-9. doi: 10.1016/j.bbrc.2011.06.093. Epub 2011 Jun 17.

DOI:10.1016/j.bbrc.2011.06.093

PMID:21708133

Abstract

AIMS

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP regulated chloride channel expressed in the apical plasma membrane of pancreatic duct cells where it plays an important role in fluid secretion. The purpose of this study was to elucidate the role of the CFTR chloride channel on ion and fluid secretion from the guinea-pig pancreas by manipulating the expression of CFTR by RNA interference or by luminal application of a CFTR selective activator, MPB91, in isolated cultured pancreatic ducts.

MATERIALS AND METHODS

Using cDNA isolated from the guinea-pig small intestine, fragments of the CFTR gene were generated by polymerase chain reaction and directly sequenced. Two different RNA duplexes for small interference RNA (siRNA) were designed from the sequence obtained. Fluid secretion from the isolated guinea-pig pancreatic ducts was measured using video-microscopy. The amount of CFTR chloride channel or AQP1 water channel expressed in pancreatic ducts was examined by immunoblotting with antibodies against CFTR or AQP1, respectively.

RESULTS

Guinea-pig CFTR consists of 1481 amino acid residues. An additional glutamine residue was found to be inserted between amino acid residues 403 and 404 of human CFTR. Forskolin-stimulated fluid secretion from intact pancreatic ducts was significantly higher in the presence of MPB91 compared to fluid secretion in the absence of MPB91. Both basal and forskolin-stimulated fluid secretion in pancreatic ducts transfected with CFTR specific siRNAs were reduced by ∼50% compared to fluid secretion from ducts transfected with scrambled negative control dsRNAs. The amount of CFTR and AQP1 proteins was reduced to 34% and 45% of control, respectively.

CONCLUSIONS

The activity of the CFTR chloride channel or the amount of CFTR protein expressed determines the rate of fluid secretion from the isolated guinea-pig pancreatic ducts.

摘要

目的

囊性纤维化跨膜电导调节因子（CFTR）是一种环磷酸腺苷调节的氯离子通道，表达于胰腺导管细胞的顶端质膜，在其中发挥着重要的分泌作用。本研究旨在通过 RNA 干扰操纵 CFTR 的表达或通过腔管内应用 CFTR 选择性激活剂 MPB91，阐明 CFTR 氯离子通道在豚鼠胰腺离子和液体分泌中的作用。

材料和方法

使用从小鼠肠道分离的 cDNA，通过聚合酶链反应生成 CFTR 基因片段，并直接测序。根据获得的序列设计了两个用于小干扰 RNA（siRNA）的不同 RNA 双链。使用视频显微镜测量分离的豚鼠胰腺导管的液体分泌量。通过针对 CFTR 或 AQP1 的抗体进行免疫印迹，分别检测胰腺导管中 CFTR 氯离子通道或 AQP1 水通道的表达量。

结果

豚鼠 CFTR 由 1481 个氨基酸残基组成。在人类 CFTR 的 403 位和 404 位氨基酸之间发现了一个额外的谷氨酰胺残基插入。与不存在 MPB91 的情况下相比，在存在 MPB91 的情况下，完整胰腺导管中forskolin 刺激的液体分泌显著增加。与用随机阴性对照 dsRNA 转染的导管相比，用 CFTR 特异性 siRNA 转染的导管中的基础和 forskolin 刺激的液体分泌均减少了约 50%。CFTR 和 AQP1 蛋白的量分别减少到对照的 34%和 45%。