Lambiase Alessandro, Micera Alessandra, Pellegrini Graziella, Merlo Daniela, Rama Paolo, De Luca Michele, Bonini Sergio, Bonini Stefano
CIR Laboratory of Ophthalmology, University Campus Bio-Medico, Rome, Italy.
Invest Ophthalmol Vis Sci. 2009 Oct;50(10):4622-30. doi: 10.1167/iovs.08-2716. Epub 2009 Apr 30.
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.
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.
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.
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.
释放到泪膜中的黏蛋白对维持健康的眼表至关重要。在慢性眼表炎症性疾病中可观察到杯状细胞数量和黏蛋白分泌的改变。神经生长因子（NGF）在眼表愈合和炎症中起关键作用。本研究的目的是在体外评估NGF对结膜杯状细胞分化以及黏蛋白产生和分泌的影响。
将人结膜上皮细胞暴露于递增浓度的NGF（1至250 ng/mL），并进行分析以量化细胞生长（MTT/Ki67/BrdU）、杯状细胞分化（PAS/MUC5AC共聚焦染色）和黏蛋白mRNA表达（实时PCR）。还分别通过夹心ELISA和FACS分析分泌型和细胞内的MUC5AC。为了证实NGF的生物学效应，对原代培养物进行相同的评估，并研究干性标志物（p63）和定向标志物（14-3-3 sigma）的变化。
在细胞培养中，NGF诱导杯状细胞数量、MUC5AC产生、储存和释放呈剂量依赖性增加。此外，在原代培养中，NGF诱导流产集落和14-3-3 sigma蛋白增加，以及p63 mRNA和蛋白减少，表明NGF对人结膜上皮有分化作用。
这些发现表明，NGF可能在导致结膜上皮分化和黏蛋白分泌的复杂机制中起作用。除了NGF在促进眼表愈合和敏感性方面的已知作用外，其对结膜杯状细胞的影响为研究NGF在干眼病中的治疗效果提供了理论依据。
