Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 2011 Mar 1;52(3):1641-9. doi: 10.1167/iovs.10-6492.
Nuclear oxidative DNA damage increases with age in human corneal endothelial cells (HCECs) and contributes to their decreased proliferative capacity. These studies investigated whether HCECs respond to this damage by upregulating their expression of oxidative stress and DNA damage-signaling genes in an age-dependent manner.
HCECs were dissected from the corneas of young (30 years and younger) and older (50 years and older) donors. Total RNA was isolated and reverse-transcribed. Oxidative stress and DNA damage-signaling gene expression were analyzed using commercial PCR-based microarrays. Western blot analyses were conducted on selected proteins to verify the microarray results. Nuclear DNA damage foci were detected in the endothelium of ex vivo corneas by immunostaining for H2AX-Ser139.
Four of 84 genes showed a statistically significant age-related difference in the expression of oxidative stress-related genes; however, Western blot analysis demonstrated an age-related increase in only 2 (cytoglobin and GPX-1) of 11 proteins tested. No age-related differences were detected in the expression of DNA damage-signaling genes. Western blot analysis of seven DNA damage-related proteins verified this finding. Intense nuclear staining of DNA damage foci was observed in nuclei within the central endothelium of older donors. Central endothelium from young donors consistently showed a low level of positive staining.
HCECs respond to age-related increases in oxidative nuclear DNA damage by forming DNA damage repair foci; however, they do not vigorously defend against or repair this damage by upregulating the expression of multiple oxidative stress or DNA damage-signaling genes.
人眼角膜内皮细胞（HCEC）的核氧化 DNA 损伤随年龄增长而增加，这导致其增殖能力下降。本研究旨在探讨 HCEC 是否会通过上调氧化应激和 DNA 损伤信号基因的表达来对此类损伤做出反应，且这种反应是否具有年龄依赖性。
从年轻（30 岁及以下）和年老（50 岁及以上）供体的角膜中分离 HCEC。提取总 RNA 并进行逆转录。使用商业 PCR 微阵列分析氧化应激和 DNA 损伤信号基因的表达。对选定的蛋白质进行 Western blot 分析以验证微阵列结果。通过免疫染色检测 H2AX-Ser139 ，检测离体角膜内皮中的核 DNA 损伤焦点。
84 个基因中有 4 个与氧化应激相关的基因表达存在统计学上的年龄相关性差异；然而，Western blot 分析仅显示 11 个测试蛋白中有 2 个（细胞球蛋白和 GPX-1）存在年龄相关性增加。未检测到 DNA 损伤信号基因表达的年龄相关性差异。对 7 种与 DNA 损伤相关的蛋白质的 Western blot 分析验证了这一发现。在老年供体的中央内皮细胞核中观察到强烈的核染色 DNA 损伤焦点。年轻供体的中央内皮细胞核始终显示低水平的阳性染色。
HCEC 通过形成 DNA 损伤修复焦点来应对与年龄相关的核氧化 DNA 损伤增加，但它们不会通过上调多种氧化应激或 DNA 损伤信号基因的表达来积极防御或修复这种损伤。
