兔角膜上皮的CFTR分泌通路与细胞内钙信号释放的关联研究

摘要

背景 研究表明角膜上皮中的囊性纤维跨膜电导调节因子(CFTR)是重要的阴离子和水分泌通道,CFTR激动剂可促进泪液分泌,对干眼的治疗具有一定的意义.曾有研究认为CFTR通路是cAMP-PKA依赖通路而没有钙信号参与.然而,升高cAMP并不能促进角膜上皮CFTR通路的分泌,钙信号在角膜上皮CFTR分泌通道中是否发挥作用尚不清楚. 目的 从生理学角度探讨CFTR在兔角膜上皮分泌功能的实现与钙信号之间的关系. 方法 采用计算机随机数字分配法将16只健康新西兰白兔随机分为2个组,每组各8只.动物全身麻醉下取双眼角膜,然后处死.角膜上皮面朝向正向电流方向置于尤氏小室,奇数组兔右眼角膜仅给予单纯ATP刺激(ATP刺激组),左眼角膜用CFTR特异性抑制剂CFTRinh-172预处理后给予ATP刺激(CFTRinh-172预处理组);偶数组兔右眼角膜用于原代角膜上皮细胞培养并采用激光扫描共焦显微镜进行单个细胞胞质内钙离子荧光强度测定,左眼角膜组织用细胞内钙离子螯合剂BMPTA/AM预处理后给予ATP刺激(BMPTA/AM预处理组),采用短路电流装置记录各组兔角膜上皮的短路电流变化. 结果 ATP刺激组、CFTRinh-172预处理组和BMPTA/AM预处理组角膜上皮电流值分别为(5.73±1.36)、(1.30±0.95)和(2.47±0.55) μA/cm2,CFTRinh-172预处理组和BMPTA/AM预处理组角膜上皮电流值均低于单纯ATP刺激组,差异均有统计学意义(t=11.201、5.508,均P＜0.001).单细胞的细胞内钙离子检测发现,ATP刺激后细胞内钙离子荧光强度迅速升高至ATP刺激前的3.25倍.结论 ATP可引起兔角膜上皮细胞分泌增加,CFTRinh-172抑制整个CFTR通路中ATP促发的角膜短路电流,而BMPTA/AM消除了细胞内游离的钙离子,提示兔角膜上皮细胞分泌的CFTR通道功能的实现与细胞内钙信号释放有关.%Background Researches showed that cystic fibrosis transmembrane conductance regulator protein(CFTR) is a channel secreting anion and water,and it plays an important role in tear secreting.Traditional conception thought that CFTR pathway is cAMP-PKA-dependent without the participation of intracellular calcium.However,studies disclosed that elevating intracellular cAMP could not open the CFTR channel.So whether calcium signal is associated with CFTR-related corneal epithelial secretion is controversial.Objective This study was to investigate the association between CFTR secretion and intracellular calcium signaling in rabbit corneal epithelium.Methods Sixteen New Zealand white rabbits were randomized into odd number group and even number group by computer randomized number method.The corneas were obtained under the general anesthesia and placed in Ussing Chamber for the record of short cricuit current (Isc).The right eyes of rabbits in the odd number group were stimulated with ATP and served as ATP stimulating group.The left eyes were pretreated with CFTRinh-172 prior to ATP stimulation and served as CFTRinh-172 pretreated group.In the even number group,the left eyes of rabbits were pretreated with BMPTA/AM before ATP stimulation and served as BMPTA/AM pretreated group,and the right eyes of the rabbits were used to isolate and culture corneal epithelial cells by explant adherent method,the level of intracellular calcium were evaluated using Leica SP5 laser scan confocal microscope.Results The ATP-induced ΔIsc of corneal epithelium was (5.73 ± 1.36),(1.30 ± 0.95) and (2.47 ± 0.55) μA/cm2 in the ATP stimulating group,CFTRinh.172 pretreated group and BMPTA/AM pretreated group,respectively,and the AIsc was significantly reduced in the CFTRinh.172 pretreated group and BMPTA/AM pretreated group compared with ATP stimulating group (t=11.201,5.508,both at P ＜ 0.001).The fluorescence intensity of intracellular calcium release after ATP stimulation was 3.25 folds more than that before ATP stimulation.Conclusions ATP promotes rapid short circuit current of corneal epithelium.CFTRinh-172 depresses the ATP-induced corneal epithelium AIsc,and BMPTA/AM suppresses intracellular calcium release.It is suggested that intracellular calcium signaling secretion probably participates in the functional CFTR activity in rabbit corneal epithelium.