In Vitro Evidence of Nerve Growth Factor Effects on Human Conjunctival Epithelial Cell Differentiation and Mucin Gene Expression
In Vitro Evidence of Nerve Growth Factor Effects on Human Conjunctival Epithelial Cell Differentiation and Mucin Gene Expression
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DOI：
10.1167/iovs.08-2716
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发表时间：
2009-10-01
影响因子：
4.4
通讯作者：
Bonini, Stefano
中科院分区：
文献类型：
--
作者：
Lambiase, Alessandro;Micera, Alessandra;Bonini, Stefano
目的。释放到泪膜中的粘蛋白对维持健康的眼表至关重要。在慢性眼表炎性疾病中观察到杯状细胞数量和粘蛋白分泌的变化。神经生长因子(NGF)在眼表的愈合和炎症中起着至关重要的作用。方法：将人结膜上皮细胞暴露于不同浓度的NGF(1~250 ng/mLNGF)中，分析其对细胞生长(MTT/Ki67/BrdU)、杯状细胞分化(PAS/MUC5AC共聚焦染色)和粘蛋白基因表达(Real-time PCR)的影响。分泌型和细胞型MUC5AC分别用双抗体夹心酶联免疫吸附试验和流式细胞仪检测。为了证实NGF的生物学效应，在原代培养上进行了同样的评估，并观察了干性(P63)和承诺(14-3-3西格玛)标志物的变化。结果：在细胞培养中，NGF诱导杯状细胞数量、MUC5AC的产生、储存和释放呈剂量依赖性增加。此外，在原代培养中，NGF诱导的流产集落和14-3-3sigma蛋白表达增加，p63mRNA和蛋白表达减少，提示NGF对人结膜上皮细胞具有分化作用。结论NGF可能在结膜上皮细胞分化和粘蛋白分泌的复杂机制中发挥作用。除了NGF在促进眼表愈合和敏感性方面的已知作用外，它对结膜杯状细胞的影响也为研究NGF对干眼病的治疗效果提供了理论基础。(投资眼科VS科学。2009年；50：4622-4630)网址：10.1167/iovs.08-2716
PURPOSE. Mucins released into the tear film are crucial to maintaining a healthy ocular surface. Alterations in goblet cell numbers and mucin secretion are observed in chronic ocular surface inflammatory diseases. Nerve growth factor (NGF) plays a crucial role in healing and inflammation of the ocular surface. The aim of this study was to evaluate in vitro the effect of NGF on conjunctival goblet cell differentiation and mucin production and secretion.METHODS. Human conjunctival epithelial cells were exposed to increasing NGF concentrations (1 to 250 ng/mL) and analyzed to quantify cell growth (MTT/Ki67/BrdU), goblet cell differentiation (PAS/MUC5AC confocal staining), and mucin mRNA expression (real-time PCR). Secreted and cellular MUC5AC were also analyzed by sandwich-ELISA and FACS, respectively. To confirm the biological effects of NGF, the same evaluations were performed on primary cultures, and changes in markers of stemness (p63) and commitment (14-3-3 sigma) were also investigated.RESULTS. In cell cultures, NGF induced a dose-dependent increase of goblet cell numbers, MUC5AC production, storage, and release. Additionally, in primary cultures, NGF induced an increase of abortive colonies and 14-3-3 sigma protein, and a decrease of p63 mRNA and protein, suggesting a differentiating effect of NGF on human conjunctival epithelium.CONCLUSIONS. These findings show that NGF might play a role in the complex mechanism leading to conjunctival epithelium differentiation and mucin secretion. In addition to the known roles of NGF in promoting ocular surface healing and sensitivity, its effects on conjunctival goblet cells support a rationale to investigate the therapeutic effectiveness of NGF in dry eye disease. (Invest Ophthalmol Vis Sci. 2009; 50: 4622-4630) DOI:10.1167/iovs.08-2716