非损伤检测胎儿肺上皮细胞微环境中Cl-流的变化情况
肺上皮细胞中体液运输由上皮细胞膜两侧渗透压产生的Na+和Cl-流调节支配，而离子转运过程中所需的能量是由活性Na+/K+ATP酶所提供，Na+和Cl-的跨膜分布是由配体调节的离子通道和交换器所控制，所以如果Cl-的转运发生缺陷将导致与体液运输失调相关的疾病。
囊性纤维病的发生就是由于Cl-转运的缺失而造成。对于这种疾病的研究，先前采用的检测方法，如同位素示踪法检测到的并不是单一的Cl-在胞内外的转运情况，而是进出细胞的所有离子转运的总体情况，所以得到的结论并不能精确的说明Cl-在细胞内外的变化情况。而非损伤微测技术（SrE）通过选用Cl-自参比电极检测胞外Cl-流的变化的方法克服了这个问题。
在该研究中通过选择性Cl-自参比电极实时、高分辨率、非损伤性的方式来检测胎儿远端肺上皮细胞基底或顶端在激动剂作用下Cl-流的变化方向和胞外Cl-数量变化。研究结果表明肺远端顶膜生理学微环境中体液转运与Cl-梯度存在密切关系，Cl-的动力学梯度能够作为质膜微环境中真实的Cl-指示者。这对于理解通气管表面液体中的盐浓度调控细胞和分子过程与上皮体液转运的关系非常重要。
关键词：自参比电极(self-referencing electrode,SrE);lung(肺);囊性纤维化(cystic Fibrosis);β2肾上腺素受体(β2 adrenoreceptor);氯离子流(Cl- flux)
参考文献：LAND SC, et al. The Journal of Experimental Biology, 2001, 204, 785-795
全文下载：http://jeb.biologists.org/cgi/reprint/204/4/785
A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (JCl) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e)
and in response to pharmacological challenges. The technique, which is
similar to that developed for other ion-selective microelectrodes,
centres on the oscillation of a Cl--selective microelectrode
between known points, micrometres apart, orthogonal to the plasma
membrane. Application of the Fick principle to the differential voltage
obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmolcm-2 s-1). A Cl- effusion gradient was used to confirm that empirical measurements of JCl using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical JCl was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50mmoll-1, an apical-tobasolateral (inward) flux, maximal at 400 pmolcm-2 s-1, was observed; this reverted to a continuous basolateralto-apical (outward) flux of 203 pmolcm-2 s-1 at [Cl]e> 100 mmoll-1. At [Cl-]e>100mmoll-1, isoproterenol (basolaterally applied, 10 mmol l-1) activated a Cl- influx of 561 pmolcm-2 s-1, whereas UTP (apically applied, 100 mmol l-1) stimulated a Cl- efflux of 300 pmolcm-2 s-1. In all cases, 50–70 % of JCl was abolished by Cl- channel blockade using 10 mmol l-1 diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl-
gradient in the microenvironment of the apical region of lung epithelia
that varies in both direction and magnitude as a function of external
[Cl-]e and in response to Cl- channel blockade and to β2 adrenoreceptor and P2Y receptor agonists.
Key words: self-referencing electrode, fluid transport, lung, cystic fibrosis, β2 adrenoreceptor, P2Y receptor, Cl- flux.
-
科技前沿
