氧化衰老、细胞外基质和光感受器外节膜盘对RPE细胞中黏着斑激酶表达的影响

Effect of H2O2,extracellular matrix and out segment of photoreceptor on expression of focal adhesion kinase in RPE cell

背景脉络膜新生血管（CNV）是引起视力障碍的重要原因之一，发生机制复杂。黏着斑激酶（FAK）在调控细胞增生、存活、移行和分化等细胞进程中发挥重要作用，参与新生血管的应答。然而，FAK在CNV发生中的作用尚未见相关报道。目的观察氧化衰老、细胞外基质（ECM）成分和吞噬光感受器外节膜盘（POS）等CNV生成相关因素作用下人视网膜色素上皮（RPE）细胞中FAK的表达及磷酸化变化，探讨FAK在CNV发生中的作用。方法体外原代培养来自于供体眼的人RPE细胞，并分别暴露于H2O2氧化衰老、吞噬POS和ECM成分等刺激因素。用10、20、50、100μmol／L4种浓度的H2O2处理培养的RPE细胞20d，诱导RPE细胞氧化衰老；用含终密度1×10^6／mlPOS的培养液培养RPE细胞20d，设置正常细胞对照组、单纯POS处理组、200μmol／LCoCl2缺氧组及POS＋缺氧组；将RPE细胞接种于涂布100mg／L的纤维连接蛋白（FN）、层黏连蛋白（LN）和I型胶原酶的培养皿中，分别继续培养30rain和1h。在不同时间点收集细胞提取总蛋白，Western blot法观察不同时间点RPE细胞中FAK的表达及磷酸化FAK（pFAK）变化。结果20μmol／L及50μamol／LH2O2处理组FAK蛋自在RPE细胞的表达量明显高于对照组，而pFAK在各H2O2处理组均较对照组明显下降，差异均有统计学意义（P〈0．01）。长期吞噬POS后，RPE细胞内出现消化不完全的溶酶体颗粒；单纯POS处理组与对照组间RPE细胞中FAK和pFAK的表达量差异无统计学意义（P〉0．05），但单纯缺氧组FAK和pFAK的表达量较对照组均升高，POS＋缺氧组pFAK的表达较单纯缺氧组显著升高，差异均有统计学意义（P〈O．01）。与对照组比较，在FN、LN和I型胶原刺激后1h，RPE细胞中pFAK表达上调，差异均有统计学意义（P〈0．05，P〈O．01）。结论在衰老、吞噬POS和ECM成分等因素作用下，RPE细胞中出现FAK的表达及磷酸化．提示FAK在CNV发生过程中发挥调控作用。

Background The underlying mechanism of choroidal neovascularization（CNV） is muhifactorial and complex. Focal adhesion kinase （ FAK ） plays a crucial role in controlling essential cellular processes and influencing distinct steps of the angiogenic response. But to our knowledge, seldom study on the effect of FAK on CNV formation has been reported previously. Objective In this study, the effect of several CNV risk factors on the expression of FAK in cultured retinal pigment epithelium（RPE） cells was investigated to illuminate effect of FAK on CNV. Methods Human RPE cells were isolated from donor eyes and exposed to H2O2, swallow of outer segment of photoreceptors（POS） and extracellular matrix （ECM） separately with the treating as follows： RPE cells were cocultured with 10,20,50 and 100μmol/L H2O2 for 20 days ; POS （ 1× 10^6/ml） were co-cultivated with RPE cells for 20 days（setting control group, POS group, hypoxia group with 200 μmol/L CoC12, and POS＋hyoxia group）;RPE cells were cultured on the plates coated with 100 mg/L fibronectin（ FN ） , laminin（LN） or collagen type I for 30 minutes or 1 hour. The expression of FAK and pFAK in RPE cells were examined by Western blot analysis. Results FAK was highly expressed in the 20 μmol/L and 50 μmol/l, H2O2 groups compared with control group （P〈O. O1 ） ;while heexpression level of pFAK was reduced after treated with n202 in comparison with the control group（ P〈0. 01 ）. After cultured with POS for 20 days,the undigested lysosome could be observed in RPE cells. The expressions of FAK and pFAK in RPE cells were not significantly changed between control group and POS groups （P〉0.05） , but those in hypoxia group were significantly up-regulated in comparison with control group（P〈0. 01 ）. Compared with the hypoxia group,the expression amount of pFAK was elevated in POS＋hyoxia group（ P〈0. 01 ）. In comparison with control group, the increased pFAK expression was seen in FN ,LN and collagen typeltreating for 1-hour groups（ P〈O. 05 ,P〈0. 01 ）. Conclusion FAK pathway participates in several CNV-initiated signaling, such as H2 02, POS and ECM,in cultured RPE cells. It is reasonable to believe that FAK potentially plays an important role in CNV-dependent disorder.