摘要
目的 观察丝氨酸蛋白酶HtrA1沉默对光损伤人视网膜色素上皮（RPE）细胞的影响。方法 体外培养人RPE细胞，取第8～12代生长良好的传代细胞用于实验。将细胞分为正常对照组、光损伤模型组。采用波长400 nm的蓝色节能灯以（2000±500） Lux持续光照RPE细胞6 h建立光损伤模型。建模后光损伤模型组再分为针对HtrA1的小干扰RNA（HtrA1 siRNA）组、阴性对照组、空白对照组。应用脂质体RNAimax将HtrA1 siRNA转染至HtrA1 siRNA组细胞；阴性对照组细胞转染非特异的阴性siRNA；空白对照组为单纯光损伤细胞。采用细胞计数试剂盒、transwell小室法及流式细胞仪检测各组细胞增生、迁徙、凋亡以及细胞周期情况；实时聚合酶链反应和蛋白免疫印迹法检测细胞中HtrA1和血管内皮生长因子A（VEGF-A）mRNA、蛋白质表达。结果 光损伤模型组细胞中HtrA1 mRNA、蛋白表达均较正常对照组细胞明显升高，差异有统计学意义（t=17.62、15.09，P＜0.05）。与阴性对照组、空白对照组比较，HtrA1 siRNA组细胞增生（t=6.37、4.52）、迁徙能力（t=9.56、12.13）、凋亡率（t=23.37、29.08）明显下降，差异有统计学意义（P＜0.05）；细胞停留在G0/G1期数目增多（t=6.24、4.93），差异有统计学意义（P＜0.05）；HtrA1、VEGF-A mRNA（t=17.36、11.32、7.29、4.05）和蛋白（t=12.02、15.28、4.98、6.24）表达均明显降低，差异有统计学意义（P＜0.05）。结论 特异性沉默HtrA1基因表达可降低蓝光对人RPE细胞增生、迁徙能力的损伤和细胞凋亡率；下调VEGF-A的表达。
Objective To observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium （RPE） cells. Methods Cultured human RPE cells（8th - 12th generations）were exposed to the blue light at the intensity of （2000 ± 500） Lux for 6 hours to establish the light injured model. Light injured cells were divided into HtrA1 siRNA group, negative control group and blank control group. HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively. The proliferation of cells was assayed by CCK-8 method. Transwell test was used to detect the invasion ability of these three groups. Flow cytometry was used to detect the cell cycle and apoptosis. The expression of HtrA1 and vascular endothelial growth factor （VEGF） -A was detected by real time-polymerase chain reaction and Western blot respectively. Results The mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells （t=17.62, 15.09; P〈0.05）. Compared with negative control group and blank control group, the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation （t=6.37, 4.52）, migration （t=9.56, 12.13）, apoptosis （t=23.37, 29.08） and decreased mRNA （t=17.36, 11.32, 7.29, 4.05） and protein levels （t=12.02, 15.28, 4.98, 6.24） of HtrA1 and VEGF-A. Cells of HtrA1 siRNA group mainly remained in G0/G1 phase, the difference was statistically significant （t=6.24, 4.93; P 〈0.05）. Conclusion Knockdown of HtrA1 gene may reduce the proliferation, migration capability and apoptosis of light-injured RPE cells, and decrease the expression of VEGF-A.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2016年第4期413-417,共5页
Chinese Journal of Ocular Fundus Diseases
基金
基金项目：国家自然科学基金（81271025、81271023）
关键词
视网膜色素上皮/病理生理学
血管内皮生长因子A
RNA干扰
丝氨酸蛋白酶类
光刺激/副作用
动物实验
Retinal pigment epithelium/physiopathology
Vascular endothelial growth factor A
RNA interference
Serine proteases
Photic stimulation/adverse effects
Animal experimentation
