急性氧化应激下器官培养中角膜内皮细胞的存活：血管活性肠肽的作用

Corneal endothelial cell survival in organ cultures under acute oxidative stress: effect of VIP.

作者信息

Koh S W, Waschek J A

机构信息

Department of Ophthalmology, University of Maryland at Baltimore, USA.

出版信息

Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4085-92.

PMID:11095600

Abstract

PURPOSE

Human corneal endothelium, a neural crest-derived tissue, has a very limited regenerative capacity and may depend on trophic factors for its survival throughout life, as well as after injury and during storage before transplantation. The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a 28-amino acid neurotrophic factor present in human aqueous humor, promotes the survival of corneal endothelium in corneal organ cultures, and whether VIP is produced by the corneal endothelium.

METHODS

Thirteen viable human donor corneas that had been received from the Central Florida Lions Eye Bank and stored in preservation medium (Optisol-GS; Chiron Vision, Irvine, CA) at 4 degrees C for 8 to 17 days were bisected. Each half was treated with either 0 or 10 nM VIP (15 minutes) and subjected to H(2)O(2) (1.4 mM, 30 minutes) treatment at 37 degrees C. The numbers of live and dead corneal endothelial (CE) cells isolated from the corneas were then determined under fluorescence microscopy using a live-dead viability-cytotoxicity assay conducted by an observer uninformed of the treatment. The effect of VIP (10(-16) to 10(-6) M) on CE cell survival was also studied in fresh bovine corneas in situ, by using the same assay. The presence of VIP in the corneal endothelium in fresh human donor and bovine eyes was examined by immunocytochemistry, in situ hybridization, and Western blot analysis, whereas VIP in the bovine aqueous humor was assessed by radioimmunoassay.

RESULTS

VIP (10 nM) significantly increased CE survival in 10 of 13 human corneas. The mean survival of CE cells (+/-SEM) was 42% +/- 3% in control corneas versus 59% +/- 3% in VIP-treated corneas (P < 0.001). In bovine corneas, VIP at concentrations as low as 10(-10) M demonstrated a significant protective effect. The mean number of dead CE cells on bovine corneas was maximally decreased by 10(-6) M VIP from 46 +/- 5 to 18 +/- 3 per field. In CE cells from fresh human and bovine corneas, VIP immunoreactivity and mRNA were detected. VIP was also present in bovine aqueous humor at 40 +/- 8 pM.

CONCLUSION

VIP may be an autocrine trophic factor that protects CE cells from H(2)O(2) in normal aqueous humor and possibly from other oxidative insults.

摘要

目的

人角膜内皮是一种神经嵴衍生组织，其再生能力非常有限，在其整个生命周期、损伤后以及移植前储存期间的存活可能依赖于营养因子。本研究的目的是确定血管活性肠肽（VIP），一种存在于人类房水中的28个氨基酸的神经营养因子，是否能促进角膜器官培养中角膜内皮的存活，以及角膜内皮是否产生VIP。

方法

从佛罗里达州中部狮子眼库获得并在4℃下于保存培养基（Optisol - GS；Chiron Vision，尔湾，加利福尼亚州）中储存8至17天的13个存活的人供体角膜被切成两半。每一半分别用0或10 nM VIP处理（15分钟），并在37℃下进行H₂O₂（1.4 mM，30分钟）处理。然后，在荧光显微镜下，由对处理不知情的观察者使用活死细胞活力 - 细胞毒性测定法确定从角膜中分离出的活的和死的角膜内皮（CE）细胞的数量。还通过相同的测定法在新鲜牛角膜原位研究了VIP（10⁻¹⁶至10⁻⁶ M）对CE细胞存活的影响。通过免疫细胞化学、原位杂交和蛋白质印迹分析检查新鲜人供体和牛眼中角膜内皮中VIP的存在，而通过放射免疫测定法评估牛房水中的VIP。

结果

在13个人角膜中的10个中，VIP（10 nM）显著提高了CE的存活率。对照角膜中CE细胞的平均存活率（±SEM）为42%±3%，而VIP处理的角膜中为59%±3%（P < 0.001）。在牛角膜中，低至10⁻¹⁰ M的VIP表现出显著的保护作用。牛角膜上死的CE细胞的平均数量通过10⁻⁶ M VIP从每视野46±5最大程度地减少到18±3。在新鲜人角膜和牛角膜的CE细胞中，检测到VIP免疫反应性和mRNA。牛房水中也存在VIP，浓度为40±8 pM。