Information on healthy and aniridia subjects.

Purpose
In congenital aniridia, not only limbal epithelial cells but also limbal stromal cells may contribute to the development of aniridia associated keratopathy (AAK). Secondary glaucoma affects 50–75% of patients with congenital aniridia, and prostaglandin analogs are commonly used for conservative treatment. This study aimed to explore the effect of travoprost on corneal limbal stromal cells from healthy (LSCs) and congenital aniridia subjects (AN-LSCs), in vitro.
Materials and methods
Cells were extracted from aniridia (AN-LSCs) (n=7) and healthy donors (LSCs) (n=7). In culture, the cells were treated with travoprost at concentrations ranging from 0.039–40 μg/mL for 20 minutes. Cell viability, proliferation and migration were determined to assess the effect of travoprost on AN-LSCs and LSCs. Analysis of inflammation-, retinoic acid signaling-, and apoptosis-related genes and proteins was performed using qPCR, Western blot, and ELISA. One-way ANOVA was used to analyze cell viability and proliferation. The Mann–Whitney test was applied to compare between-group differences, while the Friedman test was used to assess within-group differences.
Results
Both in LSCs and AN-LSCs, travoprost treatment at 0.078 μg/mL and higher concentrations significantly reduced cell viability (p≤0.033; p<0.001) and proliferation decreased both in LSCs and AN-LSCs at 40 μg/mL travoprost concentration (p=0.006; p=0.002). At 6 and 12 hours, 0.313 μg/mL travoprost significantly increased the migration rate of AN-LSCs (p=0.021; p=0.021). AN-LSCs displayed lower PAX6 and JNK (MAPK8) mRNA (p<0.001) but higher MMP-3, MMP-9, ADH7, FABP5 and VEGFA mRNA levels (p≤0.037) than LSCs. PTGFR and JNK mRNA levels, MMP9 and ADH7 protein levels increased significantly in AN-LSCs after 0.313 μg/mL travoprost treatment (p≤0.039), while NF-κB and ADH7 protein levels decreased significantly in LSCs using 0.313 μg/mL travoprost (p=0.039; p<0.001).
Conclusions
Travoprost may affect viability, proliferation, and migration of both LSCs and AN-LSCs, with AN-LSCs exhibiting greater sensitivity than LSCs. Additionally, travoprost may regulate MMP-9 expression in AN-LSCs via the JNK signaling pathway. Furthermore, in AN-LSCs, travoprost treatment does not lead to a decrease in NF-κB and ADH7 protein levels.

### 研究背景与目的
在先天性无虹膜症（congenital aniridia）中，不仅角膜缘上皮细胞，角膜缘基质细胞也可能参与无虹膜相关角膜病（aniridia associated keratopathy, AAK）的发生发展。继发性青光眼在先天性无虹膜症患者中的发生率为50%~75%，前列腺素类似物（prostaglandin analogs）常被用于保守治疗。本研究旨在体外探索曲伏前列素（travoprost）对健康受试者来源角膜缘基质细胞（limbal stromal cells, LSCs）及先天性无虹膜症患者来源角膜缘基质细胞（AN-LSCs）的作用。

### 材料与方法
本研究从7例先天性无虹膜症患者样本中分离提取得到AN-LSCs，同时从7例健康供者样本中分离提取得到LSCs。将体外培养的细胞置于浓度范围为0.039~40 μg/mL的曲伏前列素培养基中处理20分钟。通过检测细胞活力、增殖能力与迁移能力，评估曲伏前列素对AN-LSCs与LSCs的作用。采用实时荧光定量PCR（qPCR）、蛋白质印迹（Western blot）及酶联免疫吸附实验（ELISA），分析炎症相关、视黄酸信号通路相关及凋亡相关的基因与蛋白表达水平。采用单因素方差分析（one-way ANOVA）分析细胞活力与增殖数据；组间差异比较采用曼-惠特尼U检验（Mann–Whitney test），组内差异比较则采用弗里德曼检验（Friedman test）。

### 结果
在LSCs与AN-LSCs中，0.078 μg/mL及更高浓度的曲伏前列素处理均可显著降低细胞活力（p≤0.033；p<0.001）；当曲伏前列素浓度为40 μg/mL时，LSCs与AN-LSCs的增殖能力均显著下降（p=0.006；p=0.002）。在处理后6小时与12小时，0.313 μg/mL的曲伏前列素可显著提升AN-LSCs的迁移速率（p=0.021；p=0.021）。与LSCs相比，AN-LSCs的PAX6及JNK（MAPK8）mRNA表达水平显著降低（p<0.001），但MMP-3、MMP-9、ADH7、FABP5及VEGFA的mRNA表达水平显著升高（p≤0.037）。经0.313 μg/mL曲伏前列素处理后，AN-LSCs中PTGFR与JNK的mRNA表达水平，以及MMP9、ADH7的蛋白表达水平均显著上调（p≤0.039）；而LSCs经0.313 μg/mL曲伏前列素处理后，NF-κB与ADH7的蛋白表达水平显著下调（p=0.039；p<0.001）。

### 结论
曲伏前列素可影响LSCs与AN-LSCs的活力、增殖能力与迁移能力，且AN-LSCs对曲伏前列素的敏感性高于LSCs。此外，曲伏前列素可能通过JNK信号通路调控AN-LSCs中MMP-9的表达。进一步研究发现，在AN-LSCs中，曲伏前列素处理不会导致NF-κB与ADH7的蛋白表达水平下降。