Kamao Hiroyuki, Miki Atsushi, Kiryu Junichi
Department of Ophthalmology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0114, Japan.
J Ophthalmol. 2019 Jun 19;2019:9428738. doi: 10.1155/2019/9428738. eCollection 2019.
No standard therapy for RPE tear, a complication of neovascular age-related macular degeneration, exists even though RPE tears cause severe vision loss, and promotion of cell proliferation and/or migration could be a candidate RPE tear therapy. The aim of this study is to evaluate the effect of Rho-associated coiled-coil containing kinase (ROCK) inhibitor Y27632 on retinal pigment epithelial (RPE) cell motility during wound healing.
Human RPE cells were cultured in media with and without 10 M Y27632. A luminescent cell viability assay and vinculin immunocytochemistry were used to test the Y27632 effect on RPE cell adhesion. The mean size of vinculin puncta was quantified from immunofluorescence images. RPE cell motility during wound healing was evaluated using time-lapse imaging and measuring cell migration distances and cell coverage rate in wound fields.
The number of adhered RPE and mean size of vinculin puncta were, respectively, 20519 cells and 3.65 m under nontreatment and 23569 cells and 0.66 m under Y27632 treatment. Cell migration distance and cell coverage percentage for untreated and Y27632-treated cells were 98.9 and 59.4% and 203.4 and 92.5%, respectively.
Inhibition of ROCK signaling by using 10 M Y27632 promoted RPE cell motility during wound healing by reducing RPE cell adhesion strength.
尽管视网膜色素上皮（RPE）撕裂会导致严重视力丧失，但目前尚无针对新生血管性年龄相关性黄斑变性并发症RPE撕裂的标准治疗方法，促进细胞增殖和/或迁移可能是一种RPE撕裂治疗方案。本研究旨在评估Rho相关卷曲螺旋蛋白激酶（ROCK）抑制剂Y27632对伤口愈合过程中视网膜色素上皮（RPE）细胞运动的影响。
将人RPE细胞培养在含有和不含有10μM Y27632的培养基中。采用发光细胞活力测定法和纽蛋白免疫细胞化学法检测Y27632对RPE细胞黏附的影响。从免疫荧光图像中定量纽蛋白斑点的平均大小。使用延时成像技术评估伤口愈合过程中RPE细胞的运动情况，并测量伤口区域的细胞迁移距离和细胞覆盖率。
在未处理条件下，黏附的RPE细胞数量和纽蛋白斑点的平均大小分别为20519个细胞和3.65μm，在Y27632处理条件下分别为23569个细胞和0.66μm。未处理和Y27632处理的细胞的迁移距离和细胞覆盖百分比分别为98.9和59.4%以及203.4和92.5%。
使用10μM Y27632抑制ROCK信号通路可通过降低RPE细胞黏附强度来促进伤口愈合过程中RPE细胞的运动。
