Functional CNTF receptor alpha subunit restored by its recombinant in corneal endothelial cells in stored human donor corneas: connexin-43 upregulation.
Functional CNTF receptor alpha subunit restored by its recombinant in corneal endothelial cells in stored human donor corneas: connexin-43 upregulation.
复制标题
功能性 CNTF 受体 α 亚基通过其在保存的人类供体角膜的角膜内皮细胞中的重组而恢复：connexin-43 上调。
DOI：
10.1167/iovs.08-2590
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发表时间：
2009
影响因子：
4.4
通讯作者：
Ku,CYPaul
中科院分区：
文献类型：
--
作者：
Koh,Shay-WheyM;Celeste,Jordan;Ku,CYPaul
目的。睫状神经营养因子（Ciliary neurotrophic factor， CNTF）用于视网膜退化的临床试验正在进行中，然而研究表明CNTF受体结合α-亚基（CNTFRα）可从损伤组织中释放。在这里，重组人（rh） CNTFRα被证明可以恢复在角膜储存方法中失去内源性CNTFRα的人角膜内皮（CE）细胞的功能CNTFRα。在保存的人供体角膜CE细胞中，定量内源性CNTFRα水平（通过Western blot分析），证实CNTF刺激导致连接蛋白43上调，并检测rhCNTFRα (8.3 nM)增强CNTF （0.83 nM）效应的有效性。将配对的人供体角膜与CNTF处理或CNTF与（rhCNTFRα+ CNTF）处理对照（24小时，37℃），分别采用半定量RT-PCR和Western blot分析CE细胞连接蛋白-43 mRNA和蛋白。与储存的人角膜孵育90分钟后，Western blot分析结果表明rhCNTFRα与CE膜组分结合。CE细胞CNTFRα水平随角膜储存时间的增加而降低。CNTF处理和（rhCNTFRα+ CNTF）处理的配对角膜CE细胞connexin-43 mRNA水平平均（平均±SEM）分别为0.26±0.08和0.58±0.21 （P= 0.029, 8对，保存时间≥25天）。在蛋白水平上证实了rhCNTFRα的增强。在保存时间短（≤9天）且内源性CNTFRα丰富的角膜中，rhCNTFRα降低了CNTF的有效性。rhCNTFRα被纳入CE膜。rhCNTFRα作为CNTF信号中缺失的内源性膜结合CNTFRα的替代品，提示辅助rhCNTFRα治疗CNTF治疗的潜力。
purpose. Ciliary neurotrophic factor (CNTF) is undergoing testing in human clinical trials to rescue degenerating retina, whereas studies show that the CNTF-binding α-subunit of the CNTF receptor (CNTFRα) is released from injured tissues. Here, the recombinant human (rh) CNTFRα was shown to restore functional CNTFRα in human corneal endothelial (CE) cells that lost endogenous CNTFRα during corneal storagemethods. In CE cells of stored human donor corneas, endogenous CNTFRα levels were quantified (by Western blot analysis), CNTF stimulation leading to the upregulation of connexin-43 was demonstrated, and the effectiveness of rhCNTFRα (8.3 nM) in augmenting the CNTF (0.83 nM) effect was tested. Paired human donor corneas were used as vehicle versus CNTF-treated or CNTF-versus (rhCNTFRα+ CNTF)-treated (24 hours, 37 C), followed by analysis of CE cell connexin-43 mRNA and protein by semiquantitative RT-PCR and Western blot analysis, respectively. After 90-minute incubation with stored human corneas, rhCNTFRα incorporation into the CE membrane fraction was demonstrated by Western blot analysisresults. CE cell CNTFRα levels decreased as corneal storage time increased. CE cell connexin-43 mRNA levels in CNTF-treated and (rhCNTFRα+ CNTF)-treated paired corneas averaged (mean±SEM) 0.26±0.08 and 0.58±0.21, respectively (P= 0.029; eight pairs; storage time≥ 25 days). rhCNTFRα augmentation was confirmed at the protein level. In corneas with short storage times (≤ 9 days) that retained abundant endogenous CNTFRα, rhCNTFRα decreased the effectiveness of CNTF. rhCNTFRα was incorporated into CE membranesconclusions. rhCNTFRα acted as a surrogate to the lost endogenous membrane-bound CNTFRα in CNTF signaling, suggesting the potential of an adjuvant rhCNTFRα therapy in CNTF-therapy.