Department of Medicine, Sungkyunkwan University School of Medicine , Gyeonggi, Republic of Korea.
Stem Cell & Regenerative Medicine Institute, Samsung Medical Center , Seoul, Republic of Korea.
Curr Eye Res. 2020 Nov;45(11):1352-1358. doi: 10.1080/02713683.2020.1748891. Epub 2020 Apr 2.
: This study aimed to determine the effect of thymosin beta 4 (Tβ4) on human corneal epithelial cell migration and the downstream signaling pathways. Tβ4 has a role in tissue development, cell migration, inflammation, and wound healing. A previous study showed that Tβ4 directly binds to F0-F1 ATP synthase. Other studies reported the role of extracellular ATP and purinergic receptors in cell migration with several cell types. Despite advancing to the clinical stage for treatment of eye disorders, the effect of Tβ4 on human corneal epithelial cell (HCEC) migration and proliferation and the precise downstream signaling pathway(s) have not been identified. : Various concentrations of Tβ4 were tested in vitro on human corneal epithelial cell proliferation using the CCK-8 Kit and on cell migration using the gap closure migration assay. Additionally, ATP levels at various time points were determined using the ATP Lite One-Step Kit. The Fluo 8 NO Wash Calcium Assay Kit was used to measure the intracellular Ca concentration after treatment with various concentrations of Tβ4. P2X7 inhibitors were tested on ATP signaling and migration. Total- and phospho-ERK1/2 levels were determined in western blot. : Tβ4 enhanced HCEC proliferation and migration in a dose- and time-dependent manner. Moreover, these functions were related to increased extracellular ATP levels, intracellular Ca influx, and ERK1/2 phosphorylation. Tβ4-mediated HCEC migration was inhibited by specific P2X7 purinergic receptor antagonists suggesting the role of this receptor in Tβ4-mediated human corneal epithelial cell migration. : These results suggest that Tβ4-mediated HCEC proliferation and migration are associated with increased ATP levels, P2X7 R-mediated Ca influx, and the ERK1/2 signaling pathway. This study begins to describe the mechanisms for Tβ4-mediated corneal healing and regeneration.
: 本研究旨在探讨胸腺肽β 4（Tβ4）对人角膜上皮细胞迁移的影响及其下游信号通路。Tβ4 在组织发育、细胞迁移、炎症和伤口愈合中发挥作用。先前的研究表明，Tβ4 可直接与 F0-F1 ATP 合酶结合。其他研究报告了细胞外 ATP 和嘌呤能受体在几种细胞类型的细胞迁移中的作用。尽管 Tβ4 已推进到治疗眼部疾病的临床阶段，但 Tβ4 对人角膜上皮细胞（HCEC）迁移和增殖的影响以及确切的下游信号通路尚未确定。 : 在体外，使用 CCK-8 试剂盒测试了不同浓度的 Tβ4 对人角膜上皮细胞增殖的影响，使用间隙封闭迁移测定法测试了细胞迁移的影响。此外，还使用 ATP Lite One-Step Kit 测定了不同时间点的 ATP 水平。使用 Fluo 8 NO Wash Calcium Assay Kit 测定了用不同浓度的 Tβ4 处理后细胞内 Ca 浓度。在 ATP 信号和迁移中测试了 P2X7 抑制剂。通过 Western blot 测定总 ERK1/2 和磷酸化 ERK1/2 水平。 : Tβ4 以剂量和时间依赖的方式增强 HCEC 的增殖和迁移。此外，这些功能与细胞外 ATP 水平的增加、细胞内 Ca 内流和 ERK1/2 磷酸化有关。特定的 P2X7 嘌呤能受体拮抗剂抑制了 Tβ4 介导的 HCEC 迁移，表明该受体在 Tβ4 介导的人角膜上皮细胞迁移中起作用。 : 这些结果表明，Tβ4 介导的 HCEC 增殖和迁移与 ATP 水平升高、P2X7R 介导的 Ca 内流和 ERK1/2 信号通路有关。本研究开始描述 Tβ4 介导的角膜愈合和再生的机制。
