Age-Related Gene Response of Human Corneal Endothelium to Oxidative Stress and DNA Damage
Age-Related Gene Response of Human Corneal Endothelium to Oxidative Stress and DNA Damage
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DOI：
10.1167/iovs.10-6492
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发表时间：
2011-03-01
影响因子：
4.4
通讯作者：
Zhu, Cheng C.
中科院分区：
文献类型：
--
作者：
Joyce, Nancy C.;Harris, Deshea L.;Zhu, Cheng C.
目的。人类锥形内皮细胞（HCECs）的核氧化性DNA损伤随着年龄的增长而增加，并导致其增殖能力下降。这些研究调查了HCECs是否通过上调oxi的表达来应对这种损伤。胁迫和DNA损伤信号基因的年龄依赖性。从年轻（30岁及以下）和老年（50岁及以上）供者的角膜上解剖HCECs。分离总RNA并进行逆转录。氧化应激和DNA损伤信号基因表达分析使用基于商业pcr的微阵列。对选定的蛋白质进行Western blot分析以验证微阵列结果。用h2ax - ser139免疫染色法在离体角膜内皮中检测到核DNA损伤灶。84个基因中有4个在氧化应激相关基因的表达上显示出具有统计学意义的年龄相关差异；然而，Western blot分析显示，在11种测试蛋白中，只有2种（细胞珠蛋白和GPX-1）与年龄相关。在DNA损伤信号基因的表达中没有发现与年龄相关的差异。7个DNA损伤相关蛋白的Western blot分析证实了这一发现。在老年供者的中央内皮细胞核内观察到强烈的DNA损伤灶核染色。年轻供体的中央内皮细胞始终呈低水平阳性染色。HCECs通过形成DNA损伤修复灶对年龄相关的核氧化DNA损伤增加作出反应；然而，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。（中国眼科杂志，2011;52:1641-1649）DOI:10.1167/iovs.10-6492
PURPOSE. Nuclear oxidative DNA damage increases with age in human conical endothelial cells (HCECs) and contributes to their decreased proliferative capacity. These studies investigated whether HCECs respond to this damage by upregulating their expression of oxi.dative stress and DNA damage-signaling genes in an age-dependent manner.METHODS. 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 conunercial 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.RESULTS. 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.CONCLUSIONS. 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. (Invest Ophthalmol Sci. 2011;52:1641-1649) DOI:10.1167/iovs.10-6492