力刺激调节LASIK术后圆锥角膜病变过程中MMP-2/9表达的分子机制研究

The decreased biomechanical properties as well as the abnormal MMP expression and collagen degradation after LASIK would induce postoperative complications including keratoconus. In our previous study, mechanical stretch was introduced to mimick the stress environment after LASIK, which resulted in an increase of MMP-2/9 expression in corneal cells in vitro. However, its molecular mechanism is still unclear. IL-1 and integrin signal pathway as well as the downstream ERK-MAPK pathway were suggested to participate in the mechanical stretch-induced MMP-2/9 expression in corneal cells. In light of this, Flexcell 4000 system was adopted to apply mechanical stretch on corneal cells in vitro in this study. Inhibitors of MAPK pathway, siRNA-MAPK, IL-1 receptor inhibitors as well as integrin inhibitors were incubated with corneal cells respectively to block the corresponding signal pathway. Mechanical stretch was then applied to the treated corneal cells. To analyze the effects of these pathways on the mechanical stretch-regulated MMP-2/9 activity, gene expression, protein content and activity of MMP-2/9 and its related proteins in the pathways were detected using real-time PCR, western blot and gelatin zymography. Our results can serve as the basis for better understanding the mechanism of keratoconus progression after LASIK.

LASIK术后角膜受力发生变化，MMP表达异常，胶原降解改变，角膜力学性能降低进而诱发圆锥角膜。机械拉伸能够诱导角膜细胞中MMP-2/9的表达，但其分子机制还不清楚。前期研究推测力刺激通过IL-1和整合素途径及下游的ERK-MAPK信号通路调控角膜细胞MMP-2/9的表达。本项目拟采用Flexcell 4000拉伸系统对体外培养的角膜细胞进行力刺激，用MAPK通路抑制剂、siRNA-MAPK、IL-1受体抑制剂、整合素抑制剂对角膜细胞相关信号途径进行干扰，结合力学加载，分别采用定量PCR、Western blot、明胶酶谱法检测处理前后角膜细胞中MMP-2/9相关基因表达及蛋白活性的变化，分析整合素、IL-1信号途径以及MAPK信号通路对力学加载诱导MMP-2/9基因表达的影响，初步阐明力刺激诱导角膜细胞MMP-2/9表达的分子机制，为研究圆锥角膜等LASIK术后并发症的发生机理提供参考。

LASIK术后角膜力学性能发生改变，可能引发圆锥角膜等术后并发症，MMP-2在此过程中发挥重要的作用。机械拉伸模拟术后角膜受力环境能够诱导角膜细胞中MMP-2的表达，但其分子机制还不清楚。本项目采用FLEXCELL-4000拉伸系统对体外培养的角膜细胞进行力学加载，采用基因芯片、Real-time PCR、Western blot、明胶酶谱、信号通路抑制剂结合生物信息学方法对人角膜细胞响应机械拉伸的相关信号途径进行分析，探讨了力刺激诱导角膜细胞中MMP-2表达的分子机制。主要研究结果如下：① MMP-2是角膜细胞中发挥主要的作用的明胶酶基因；② TIMP-3作为角膜细胞中MMP-2的主要内源抑制剂发挥作用，MMP-14与MMP-2的表达呈正相关；③ 筛选获得机械拉伸处理后角膜细胞中差异表达基因840条，其中表达上调基因493条，下调基因388条；机械拉伸能够诱导人角膜细胞中整合素（α-8、α-11、β3）、生长因子（IGF-2、FGF-18、VEGFA）、白介素（IL-8、IL-6）的表达。④ ERK、NF-κB信号通路抑制剂能够抑制机械拉伸诱导的MMP-2的表达。综合上述研究结果，推测角膜细胞中机械拉伸诱导MMP-2表达的信号通路：作用早期，机械拉伸能够诱导IL-8/IL-6的表达，进而激活下游的ERK-NF-κB信号途径，诱导MMP-2的表达；随着作用时间的延长，机械拉伸还可以通过整合素→生长因子→ERK信号通路调控MMP-2的表达。此外，筛选获得机械拉伸处理后差异表达的miRNA 25条，并构建差异miRNA与表达谱协同作用网络，结果推测机械拉伸通过调控miR-4521和miR-29b-1-5p的表达诱导角膜细胞中MMP-2的活性。研究结果可为进一步阐明圆锥角膜的发病机制及其临床诊断和治疗提供理论参考。