Johnson L G, Boyles S E, Wilson J, Boucher R C
Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill 27599-7020.
J Clin Invest. 1995 Mar;95(3):1377-82. doi: 10.1172/JCI117789.
Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.
囊性纤维化气道上皮细胞表现出一系列与正常情况不同的离子转运特性，不仅包括有缺陷的cAMP介导的氯离子分泌，还包括增加的钠离子吸收和增加的钙离子介导的氯离子分泌。在本研究中，我们检测了腺病毒介导的（Ad5）CFTR转导是否能纠正所有这些囊性纤维化离子转运异常。将人囊性纤维化和正常鼻上皮细胞的极化原代培养物用感染复数（moi）为10⁴的Ad5-CBCFTR感染，该感染复数几乎能转导所有细胞，或者用Ad5-CMV lacZ作为对照。与先前报道一致，Ad5-CBCFTR而非Ad5-CMV lacZ纠正了有缺陷的囊性纤维化cAMP介导的氯离子分泌。Ad5-CBCFTR（-36.9±4.8μA/cm²）将囊性纤维化气道上皮片层的基础钠离子转运速率（基础Ieq）（-78.5±9.8μA/cm²）降低到正常上皮片层测量的水平（-30.0±2.0μA/cm²），但Ad5-CMV lacZ（-65.8±6.1μA/cm²）则未降低。令人惊讶的是，与未感染的囊性纤维化上皮片层（-76.2±15.1μA/cm²）相比，在表达CFTR（已纠正）的囊性纤维化上皮片层（-6.9±11.8μA/cm²）中观察到对离子霉素（一种钙离子介导的氯离子分泌的指标）反应的Ieq显著降低。Ad5-CBCFTR对基础钠离子转运速率和钙离子介导的氯离子分泌的剂量反应效应表明，CFTR对这两种离子转运功能的调节机制可能不同。总之，CFTR的有效转导纠正了原发性囊性纤维化气道上皮细胞中钠离子的过度吸收，并将钙离子介导的氯离子分泌恢复到正常气道上皮细胞中观察到的水平。此外，评估这些离子转运异常可能代表测试囊性纤维化基因治疗疗效的重要终点。
