Unit on Vascular Diabetic Complications, Ophthalmology, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden.
PLoS One. 2013 Aug 1;8(8):e70465. doi: 10.1371/journal.pone.0070465. Print 2013.
Sight-threatening diabetic retinopathy has been treated with photocoagulation for decades but the mechanisms behind the beneficial clinical effects are poorly understood. One target of irradiation and a potential player in this process is the retinal pigment epithelium (RPE). Here we establish an in vitro model for photocoagulation of human RPE cells.
ARPE-19 cells were exposed to photocoagulation and studied at various time points up to 168h. Lesion morphology, necrosis and apoptosis were investigated by light microscopy; LIVE/DEAD staining and measurements of lactate dehydrogenase activity; and TUNEL- and ELISA-based quantification of DNA fragments, respectively. Cell migration and proliferation were explored using docetaxel and mitomycin C; temporal and spatial changes in proliferation were assessed by confocal immunofluorescence of proliferating cell nuclear antigen. Gene expression was measured by qPCR.
Photocoagulation of ARPE-19 resulted in denaturation of proteins and reproducible lesion formation. A transient peak in necrosis, followed by a peak in apoptosis was observed in cells within the lesions at 6h and 24h, respectively after photocoagulation. Cell proliferation was depressed during the first hours after photocoagulation, back to control levels at 24h and augmented in the following days. These effects were not limited to cells in the lesions, but also evident in neighbouring cells. Changes in cell proliferation during lesion repair were preceded by changes in cell migration. Altered mRNA expression of genes previously implicated in the regulation of cell proliferation (FOS, IL-1β, IL-8, HMGA2), migration and tissue repairing (TGFBR2, ADAMTS6, TIMP3, CTGF) was observed, as well as increased expression of the alarmin IL33 and the cytoprotective gene HSPA6.
Using a laser system and experimental settings that comply with standards used in clinical practice, we have established a suitable model for in vitro photocoagulation of human RPE cells to isolate their contribution to the beneficial effects of laser treatment.
几十年来，激光光凝术一直用于治疗威胁视力的糖尿病性视网膜病变，但人们对其临床疗效的潜在机制仍知之甚少。激光辐射的一个靶点和这一过程中的一个潜在参与者是视网膜色素上皮（RPE）。在此，我们建立了体外人 RPE 细胞光凝模型。
将 ARPE-19 细胞暴露于光凝，并在多达 168 小时的不同时间点进行研究。通过光镜观察损伤形态、坏死和凋亡；通过 LIVE/DEAD 染色和乳酸脱氢酶活性测量；以及 TUNEL 和 ELISA 定量检测 DNA 片段，分别检测。使用多西紫杉醇和丝裂霉素 C 研究细胞迁移和增殖；通过增殖细胞核抗原的共聚焦免疫荧光评估时间和空间上的增殖变化。通过 qPCR 测量基因表达。
ARPE-19 的光凝导致蛋白质变性和可重复的损伤形成。光凝后 6h 和 24h 分别观察到损伤内细胞出现短暂的坏死高峰和凋亡高峰。光凝后最初几小时细胞增殖受到抑制，24h 时恢复至对照水平，随后几天增加。这些影响不仅限于损伤内的细胞，也可见于邻近细胞。损伤修复过程中的细胞增殖变化先于细胞迁移变化。观察到先前涉及细胞增殖（FOS、IL-1β、IL-8、HMGA2）、迁移和组织修复（TGFBR2、ADAMTS6、TIMP3、CTGF）的基因表达改变，以及警报素 IL33 和细胞保护基因 HSPA6 的表达增加。
我们使用符合临床实践标准的激光系统和实验设置，建立了一种适合体外人 RPE 细胞光凝的模型，以分离其对激光治疗有益效果的贡献。
