a Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , University of Dublin , Dublin , Ireland.
b Trinity Centre for Bioengineering, Trinity Biomedical Science Institute, Trinity College Dublin , University of Dublin , Dublin , Ireland.
Curr Eye Res. 2019 Feb;44(2):135-146. doi: 10.1080/02713683.2018.1536216. Epub 2018 Oct 29.
To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration.
Proliferation was assessed by PrestoBlue, morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay.
Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-β1) and 3 (TGF-β3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression.
Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.
在血清补充培养基中扩增时，鉴定可促进人角膜基质细胞向成纤维细胞表型转化的生化线索，同时研究其对细胞增殖和迁移的影响。
通过 PrestoBlue 评估增殖，通过相差显微镜监测形态，通过实时聚合酶链反应（qPCR）、免疫化学和流式细胞术分析表型，通过划痕实验研究迁移。
抗坏血酸（AA）、维甲酸（RA）、胰岛素-转铁蛋白-硒（ITS）、胰岛素样生长因子 1（IGF-1）和 3-异丁基-1-甲基黄嘌呤（IBMX）促进树突状形态，增加角蛋白细胞标志物如角膜蛋白聚糖、醛脱氢酶 3 家族成员 A1（ALDH3A1）和 CD34 的表达，并防止成肌纤维细胞分化，同时在某些情况下增加增殖。转化生长因子β 1（TGF-β1）和 3（TGF-β3）促进向成肌纤维细胞分化，α-SMA 表达增加。成纤维细胞生长因子 2（FGF-2）支持成纤维细胞表型，而血小板衍生生长因子同源二聚体 B（PDGF-BB）诱导促迁移成纤维细胞表型。还将所有促角蛋白细胞因子的组合与无血清培养基进行了比较，这显著增加了 CD34 和角膜蛋白聚糖的表达。
通过去除血清并在基础培养基中添加 AA、IGF-1、RA、ITS 和 IBMX，部分恢复到静止的角蛋白细胞样表型。这些发现可用于开发基于细胞的角膜治疗方法，并在体外研究角膜疾病。
