MMP-2和FAK在单疱病毒性角膜炎中的表达

Expression of Matrix Metallo Proteinase and Focal Adhesion Kinase in Herpes Stromal Keratitis

目的:探讨Ⅰ型单纯疱疹病毒(HSV-Ⅰ)感染BALB/c小鼠眼角膜及体外培养的人角膜上皮细胞所致的基质金属蛋白酶2(MMP-2)和黏着斑激酶(FAK)表达的变化。方法:以HSV-Ⅰ感染BALB/c小鼠眼角膜制作单疱病毒性角膜炎(HSK)模型,应用免疫组织化学法检测的FAK表达;以HSV-Ⅰ感染体外培养人角膜上皮细胞(HCE),应用聚合酶链反应法、免疫印迹法、免疫组织化学法、免疫荧光法分别于2,20,40h检测HCE中MMP-2和FAK及磷酸化黏着斑激酶(phosphor-FAK)的表达情况。结果:HSK模型的角膜上皮细胞排列紊乱,基质水肿,中性粒细胞增多,FAK阳性细胞数增多,同阴性对照组相比差异具有统计学意义(P<0.05);HCE感染HSV-Ⅰ后2,20,40h,MMP-2和FAK mRNA表达分别同阴性对照组相比显著增强(P<0.01,P<0.05);感染后2hMMP-2、FAK、P-FAK蛋白表达同阴性对照组相比无显著差别(P>0.05);感染后20h、40h,MMP-2、FAK、P-FAK蛋白表达同阴性对照组相比差别显著(P<0.05)。结论:在HSV-Ⅰ感染的早期,磷酸化的FAK在病毒入侵细胞和MMP-2的激活过程中起着重要作用。FAK激活MMP-2的具体通路有待进一步研究。

Objective：To investigate the relationship between matrix metallo proteinase-2（MMP-2）and activation of focal adhesion kinase（FAK）signaling pathway in herpes stromal keratitis（HSK）.Methods：The cornea of 24BALB/c mice was infected with HSV-1to construct a model of HSK.Six additional mice served as negative controls.Immunohistochemical staining was used to detect FAK expression.Human corneal epithelium（HCE）cells cultured in vitro were infected with HSV-1,and expression of MMP-2,FAK,and phosphorylated FAK（P-FAK）in HCE were detected using reverse transcription polymerase chain reaction（RT-PCR）,Western blotting,and immunohistochemistry at the end of 2,20 and 40hours after infection.Results：In the HSK model,the corneal epithelial cells were deranged and the number of neutrophils and FAK-positive cells were increased;all were significantly different from negative controls（P〈0.05）.RT-PCR showed no significant difference in MMP-2and FAK mRNA expression in infected cells at different times,but it showed significant difference between infected cells and negative control group.There was no interaction between groups and time points.Pairwise comparisons showed that MMP-2and FAK mRNA expression were significantly increased in virus-infected cells as com-pared with controls.Over time,MMP-2and FAK mRNA expression did not differ significantly in virus-infected cells or in control cells.Western blotting assays showed no significant difference in P-FAK,FAK,or MMP-2expression between infected cells and control cells after 2h（P〈0.05）.Infected cells differed significantly from the control cells at 20 and 40h（P〈0.05）.PFAK,FAK,and MMP-2expression in virus-infected cells at 2hdiffered significantly from that respectively at 20 and 40h（P〈0.05）.Immunohistochemical staining results showed that longer infection time was associated with an increased number of cells staining positive for MMP-2,FAK,and P-FAK.Conclusion：After HSV-1infection of the corneal epithelium,the FAK signaling pathway is activated,leading to increased secretion of MMP-2in the corneal tissue and accelerated formation of corneal ulcers and necrotic lesions.