The Effects of Retinoic Acid on Human Corneal Stromal Keratocytes Cultured In Vitro Under Serum-Free Conditions
The Effects of Retinoic Acid on Human Corneal Stromal Keratocytes Cultured In Vitro Under Serum-Free Conditions
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DOI：
10.1167/iovs.13-13092
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发表时间：
2013-11-01
影响因子：
4.4
通讯作者：
Connon, Che John
中科院分区：
文献类型：
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作者：
Gouveia, Ricardo Martins;Connon, Che John
目的。视黄酸（RA）是维生素a的代谢物，在人眼的发育和功能中起着至关重要的作用。本研究的目的是探讨RA对体外无血清条件下维持较长时间的角膜基质角质细胞表型的影响。从人角膜分离的角化细胞在添加RA或DMSO载体的无血清培养基中培养21天。观察RA及其去除对细胞增殖和形态的影响。此外，分别通过定量PCR和免疫印迹法或ELISA法在转录和蛋白水平上定量检测角化细胞标志物的表达。此外，用划痕法检测RA对角化细胞迁移的影响。RA浓度达到10 × 10(-6) M时，角质细胞增殖和分层增强，流动性降低。RA还促进了角质细胞特征蛋白聚糖（如keratocan、lumican和decorin）的表达，并增加了培养物中i型胶原的数量，同时显著降低了基质金属蛋白酶1、3和9的表达。RA效应是可逆的，从培养基中去除RA后，细胞表型恢复到对照组。维甲酸通过调节细胞行为和细胞外基质组成来控制体外培养的人角膜角质细胞的表型。这些发现有助于我们理解角膜基质生物学在健康和疾病中的作用，并可能在优化角质细胞培养在组织工程、细胞生物学和医学中的应用方面证明是有用的。
PURPOSE. Retinoic acid (RA) is a metabolite of vitamin A that plays a fundamental role in the development and function of the human eye. The purpose of this study was to investigate the effects of RA on the phenotype of corneal stromal keratocytes maintained in vitro for extended periods under serum-free conditions.METHODS. Keratocytes isolated from human corneas were cultured up to 21 days in serum-free media supplemented with RA or DMSO vehicle. The effects of RA and of its removal after treatment on cell proliferation and morphology were evaluated. In addition, the expression of keratocyte markers was quantified at the transcriptional and protein levels by quantitative PCR and immunoblotting or ELISA, respectively. Furthermore, the effects of RA on keratocyte migration were tested using scratch assays.RESULTS. Keratocytes cultured with RA up to 10 x 10(-6) M showed enhanced proliferation and stratification, and reduced mobility. RA also promoted the expression of keratocyte-characteristic proteoglycans, such as keratocan, lumican, and decorin, and increased the amounts of collagen type-I in culture while significantly reducing the expression of matrix metalloproteases 1, 3, and 9. RA effects were reversible, and cell phenotype reverted to that of control after removal of RA from media.CONCLUSIONS. Retinoic acid was shown to control the phenotype of human corneal keratocytes cultured in vitro by regulating cell behavior and extracellular matrix composition. These findings contribute to our understanding of corneal stromal biology in health and disease, and may prove useful in optimizing keratocyte cultures for applications in tissue engineering, cell biology, and medicine.