Medical College, Qingdao University, Qingdao, China.
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China.
Invest Ophthalmol Vis Sci. 2019 Aug 1;60(10):3669-3679. doi: 10.1167/iovs.19-26983.
To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation.
An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and α-smooth muscle actin (α-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated β-galactosidase (SA-β-gal) staining and P16Ink4a expression. Mitogenic response and gene expression were compared among normal fibroblasts, H2O2-induced senescent fibroblasts, and TGF-β-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP).
The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-β-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16Ink4a+ vimentin+ α-SMA+, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-β-induced myofibroblasts, H2O2-induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP)1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models.
Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.
研究在涉及基质细胞凋亡、增殖和分化的角膜伤口愈合的动态过程中，成纤维细胞衰老的存在和作用。
使用 C57BL/6 小鼠的上皮清创术建立体内角膜伤口愈合模型。使用 TUNEL、Ki67 和 α-平滑肌肌动蛋白（α-SMA）分别作为凋亡、增殖和肌成纤维细胞分化的标志物对角膜进行染色。通过衰老相关β-半乳糖苷酶（SA-β-半乳糖苷）染色和 P16Ink4a 表达来确认细胞衰老。比较正常成纤维细胞、H2O2 诱导的衰老成纤维细胞和 TGF-β 诱导的肌成纤维细胞在体外的有丝分裂反应和基因表达。在角膜瘢痕、碱烧伤和穿透性角膜移植（PKP）的小鼠模型中进一步检测衰老。
上皮清创术后 4 小时和 24 小时发现角膜基质细胞的凋亡和增殖达到峰值。在 3 至 5 天，在前基质细胞中可清晰观察到 SA-β-半乳糖苷的阳性染色。衰老细胞呈 P16Ink4a+波形蛋白+α-SMA+，代表激活的角膜固有成纤维细胞的主要来源。与正常成纤维细胞和 TGF-β 诱导的肌成纤维细胞相比，H2O2 诱导的衰老成纤维细胞表现出非纤维生成表型，包括对生长因子碱性成纤维细胞生长因子（bFGF）或血小板衍生生长因子-BB（PDGF-BB）的反应性降低、基质金属蛋白酶（MMP）1/3/13 表达增加以及纤连蛋白和 I 型胶原表达减少。此外，在小鼠角膜瘢痕、碱烧伤和 PKP 模型中普遍存在细胞衰老。
角膜上皮清创术后，凋亡和增殖诱导角膜成纤维细胞衰老。衰老细胞表现出非纤维生成表型，可能参与角膜纤维化的自我限制。
