机械拉伸对人角膜基质细胞水通道蛋白AQP1表达的影响

Effect of Mechanical Stretch on the Expression of Aquaporin AQP1 in Human Keratocytes

通过探讨机械拉伸对人角膜基质细胞中水通道蛋白1(AQP1)及下游信号通路相关基因表达的影响,为揭示圆锥角膜等术后并发症的发生机制提供参考。采用FLEX-4000对人角膜基质细胞进行周期性机械拉伸处理,采用Fluo-3荧光探针检测细胞内Ca^2+的浓度,ELISA法检测拉伸处理后胞内cAMP的含量,Real-time PCR分析拉伸和Ca^2+螯合剂BAPTA处理后AQP1及其下游信号通路相关基因的表达变化。结果发现:机械拉伸可诱导角膜基质细胞中AQP1及其下游FAK、Wnt通路相关基因的表达。机械拉伸处理后胞内Ca^2+、cAMP含量显著上升。BAPTA能够抑制拉伸诱导的AQP1及下游ARHGEF2、Wnt11、MMP2基因的表达。结果提示机械拉伸能够通过调节胞内Ca^2+浓度和cAMP浓度促进AQP1的表达,并进一步通过下游的FAK、Wnt信号通路诱导MMP2的表达,参与角膜细胞外基质代谢的调节。

Mechanical stimulation plays an important role in the process of keratoconus after LASIK.However,the mechanism is still unclear.In this study,the effects of mechanical stretch on the expression of aquaporin 1(AQP1)and its downstream pathways were explored in human keratocytes,which would provide a reference for further elucidating the mechanism of keratoconus.Human keratocytes were subjected to Ca^2+chelator BAPTA treatment,or cyclic mechanical stretch with FLEX-4000.The intracellular Ca^2+concentration and cAMP content were detected by Fluo-3 fluorescent probe and ELISA respectively.The expression of AQP1 and the related genes in the downstream signaling pathway was analyzed by real-time PCR.The results show that the expression of AQP1 and its downstream genes in FAK and Wnt pathways was induced by mechanical stretch in human keratocytes.Besides,the intracellular Ca^2+and cAMP contents increased significantly after mechanical stretch.BAPTA could inhibit the stretch-induced expressions of AQP1 and its downstream genes including ARHGEF2,Wnt11,and MMP2.The results indicate that mechanical stretch could induce the expression of AQP1 in human keratocytes by regulating intracellular Ca^2+and cAMP contents,and further participate in the regulation of MMP2-related extracellular matrix metabolism through FAK and Wnt pathways.