USE OF A MEMBRANE POTENTIAL-SENSITIVE PROBE TO ASSESS BIOLOGICAL EXPRESSION OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
USE OF A MEMBRANE POTENTIAL-SENSITIVE PROBE TO ASSESS BIOLOGICAL EXPRESSION OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
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DOI：
10.1089/hum.1995.6.10-1275
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发表时间：
1995-10-01
影响因子：
4.2
通讯作者：
CABRINI, G
中科院分区：
文献类型：
--
作者：
RENIER, M;TAMANINI, A;CABRINI, G
囊性纤维化是由于一种称为囊性纤维化跨膜电导调节因子(CFTR)的氯离子转运蛋白的缺陷引起的，本研究提出了一种新的方法来评估功能性CFTR的表达，该技术使用电位敏感的探针双-(1，3-二乙硫代巴比妥酸)三亚甲基恶酚或DiSBAC(2)(3)，通过单细胞荧光成像。DiSBAC(2)(3)方法首先在小鼠乳腺肿瘤细胞系C127上得到验证，稳定表达野生型CFTR，cAMP通透性类似物8-溴-cAMP诱导的细胞膜去极化与野生型CFTR的表达一致，DiSBAC(2)(3)方法快速、简单、重复性好，不需要侵入性细胞加载程序。然后将该系统应用于人肺肿瘤细胞系A549细胞模型中，通过感染复制缺陷型腺病毒AdCFTR表达外源性CFTR，DiSBAC(2)(3)能够通过免疫细胞化学检测到CFTR蛋白表达的细胞比例，DiSBAC(2)(3)探针也用于体外培养的人鼻呼吸道细胞，它有效地区分了正常细胞和CF细胞中的内源性CFTR，所述方法对CFTR功能的评价可作为检测腺病毒载体转导的CF基因表达的有用工具，用于基因治疗试验。
Cystic fibrosis is caused by defects in a chloride-transporting protein termed cystic fibrosis transmembrane conductance regulator (CFTR), This study presents an innovative procedure to evaluate expression of functional CFTR, The technique uses the potential-sensitive probe bis-(1,3-diethylthiobarbituric acid) trimethine oxonol or DiSBAC(2)(3), by single-cell fluorescence imaging. The DiSBAC(2)(3) method was first validated on the mouse mammary tumor cell line C127, stably expressing wild-type CFTR, Activation of protein kinase A by the cAMP-permeable analogue 8-Br-cAMP induced cell membrane depolarization consistent with expression of wild-type CFTR, The DiSBAC(2)(3) method is quick, simple, and reproducible, and does not require invasive cell loading procedures. The system was then applied to the cell model of the human lung tumor cell line A549, in which exogenous CFTR was expressed by infecting with the replication-deficient recombinant adenovirus AdCFTR, DiSBAC(2)(3) was able to detect the fraction of cells in which the expression of CFTR protein was confirmed by immunocytochemistry, The DiSBAC(2)(3) probe was also used in human nasal respiratory cells cultured in vitro, in which it efficiently discriminated between endogenous CFTR in normal and CF cells, Functional evaluation of CFTR function by the described method can be a useful tool to detect the expression of the CF gene transferred by adenoviral vectors for use in gene therapy trials.