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Protection of the cornea from UVB radiation by elevated potassium in tears

通过提高泪液中的钾含量来保护角膜免受 UVB 辐射

基本信息

批准号：
8574193
负责人：
JOHN L UBELS
金额：
$ 37.59万
依托单位：
CALVIN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2017-09-29
项目状态：
已结题

项目摘要

Project summary The tears, for the most part, have an electrolyte composition like other extracellular fluids. The tears are unique, however, in that the K+ concentration ([K+] is about 20-25 mM, compared to about 5 mM in other extracellular fluids. Exposure to ultraviolet radiation causes apoptosis of cells. UVB radiation at doses relevant to ambient levels of outdoor UVB exposure activates K+ channels leading to rapid loss of intracellular K+ (K+i). We have shown that incubation of corneal epithelial cells in medium with 25 mM K+ after exposure to UVB prevents loss of K+i and inhibits activation of apoptotic mechanisms. These observations support our overall hypothesis that the high concentration of potassium ions in tears contributes to the protection of the corneal epithelium from ambient UVB-induced damage by reducing the loss of intracellular K+ when channels are activated by UVB. We now propose to investigate the intracellular signaling mechanisms by which UVB activates K+ channels and apoptotic signaling in corneal epithelial cells. The specific aims of the proposed research are: 1) to elucidate the relationships between Fas ligand- independent, UVB-induced activation of apoptotic signaling pathways and activation of K+ channels. This will be accomplished by studying the effects of UVB on apoptotic pathways and K+ channel activation in corneal epithelial cells after siRNA knockdown of Fas and FADD, or inhibition of caspase-8; 2) To investigate whether UVB-induced K+ channel activation is the primary event in activation of apoptotic pathways. K+ channels in corneal epithelial cells will be identified using a PCR array. siRNA will then be used to knock down specific channels. This will be followed by investigation of UVB-induced activation of apoptosis, as well as effects of UVB on K+ currents and intracellular [K+] when specific channels have been knocked down; 3) Using an isolated pig eye model, effects of UVB on the intact corneal epithelium will be investigated in a configuration that allows for superfusion of the ocular surface with physiologic saline in a manner that models the bathing of the cornea with tears. The hypothesis will be tested that elevated [K+] will protect the superficial corneal cell layers from UV-induced apoptosis.

项目总结与其他细胞外液一样，泪液在很大程度上含有电解质成分。泪水然而，它们的独特之处在于K+浓度([K+]约为20-25 mm，而在其他地方约为5 mm 细胞外液。暴露在紫外线辐射下会导致细胞凋亡。UVB辐射的剂量与室外UVB暴露相关的环境水平激活K+通道，导致细胞内快速丢失 K+(K+I)。我们已经证明，角膜上皮细胞在含有25 mM K+的培养液中孵育后， UVB可防止K+i的丢失，抑制细胞凋亡机制的激活。这些观察结果支持我们的总体而言，假设泪水中高浓度的钾离子有助于保护减少细胞内K+通道丢失对环境中UVB致角膜上皮损伤的影响是由UVB激活的。我们现在建议研究UVB通过的细胞内信号机制激活角膜上皮细胞的K+通道和凋亡信号。这项研究的具体目的是：1)阐明Fas配体- UVB诱导的独立的凋亡信号通路的激活和K+通道的激活。这将是通过研究UVB对角膜细胞凋亡通路和K+通道激活的影响来实现这一点 SiRNA下调Fas和FADD，或抑制caspase-8后的上皮细胞；2)研究 UVB诱导的K+通道激活是细胞凋亡途径激活的首要事件。K+通道中角膜上皮细胞将使用聚合酶链式反应阵列进行鉴定。然后，siRNA将被用来敲除特定的频道。接下来将研究UVB诱导的细胞凋亡的激活，以及当特定通道被破坏时，UVB对K+电流和细胞内[K+]的影响；3)使用建立猪眼模型，观察UVB对完整角膜上皮细胞的影响。这允许以模拟洗澡的方式向眼表面灌流生理盐水泪流满面的眼角膜。这一假设将得到检验，即升高的[K+]将保护浅层角膜来自紫外线诱导的细胞层的凋亡。