Department of Ophthalmology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Zhejiang, Hangzhou, China.
Invest Ophthalmol Vis Sci. 2011 Mar 25;52(3):1723-34. doi: 10.1167/iovs.10-5866. Print 2011 Mar.
Intracellular reactive oxygen species have been reported to associate with growth factor and integrin signalings in promoting cell adhesion in many cell types. This study is to explore if exogenous H(2)O(2) at low levels can be beneficial to cell adhesion, migration, and wound healing.
Primary rabbit corneal epithelial cells treated with 0-70 μM H(2)O(2) were tested for viability by MTT assay, adhesion by centrifugation assay, focal contacts of vinculin and F-actin by immunofluorescence, activated Src(pY416), EGF receptor (pY845), vinculin(pY1065), FAK(pY397), and FAK(pY576) by immunoblotting. Cell migration was examined with 0-50 μM H(2)O(2) using the scratch wound technique. Corneal wound healing of ex vivo pig model and in vivo mouse model was examined using H(2)O(2) with and without antioxidant N-acetylcysteine (NAC).
Compared with the untreated control, H(2)O(2) at 10-50 μM stimulated cell viability and facilitated adhesion and migration with clear induction of vinculin-rich focal adhesions and F-actin-containing stress fibers by increasing activated Src, FAK(pY576), and vinculin(pY1065). H(2)O(2) also increased phosphorylation of EGFR(Y845) parallel to that of activated Src, but both were eliminated by NAC and PP1 (Src inhibitor). Finally, H(2)O(2) induced faster wound healing in cornea both in vitro and in vivo, but the healing was diminished by NAC.
These findings suggest that H(2)O(2) at low levels promotes cell adhesion, migration, and wound healing in cornea cells or tissue, and the interaction of H(2)O(2) with Src plays a major role.
已有研究报道，细胞内的活性氧与生长因子和整合素信号有关，在促进多种细胞类型的细胞黏附中发挥作用。本研究旨在探讨低浓度的外源性 H(2)O(2) 是否有益于细胞黏附、迁移和伤口愈合。
用 MTT 法检测 0-70 μM H(2)O(2) 处理的原代兔角膜上皮细胞的活力，用离心试验检测细胞黏附，用免疫荧光法检测黏着斑处的 vinculin 和 F-actin，用免疫印迹法检测激活的Src(pY416)、表皮生长因子受体(pY845)、vinculin(pY1065)、FAK(pY397)和 FAK(pY576)。用划痕试验检测 0-50 μM H(2)O(2) 对细胞迁移的影响。用 H(2)O(2) 联合或不联合抗氧化剂 N-乙酰半胱氨酸（NAC）处理离体猪模型和体内小鼠模型，检测角膜伤口愈合情况。
与未处理对照组相比，10-50 μM 的 H(2)O(2) 刺激细胞活力，并通过增加激活的 Src、FAK(pY576)和 vinculin(pY1065)，促进富含 vinculin 的黏着斑和含有 F-actin 的应力纤维的形成，从而促进黏附和迁移。H(2)O(2) 还增加了 EGFR(Y845)的磷酸化，与激活的 Src 的磷酸化平行，但这两种作用都被 NAC 和 PP1（Src 抑制剂）消除。最后，H(2)O(2) 在体外和体内均能加速角膜伤口愈合，但 NAC 会减弱这种愈合作用。
这些发现表明，低浓度的 H(2)O(2) 促进角膜细胞或组织的黏附、迁移和伤口愈合，H(2)O(2) 与 Src 的相互作用起主要作用。
