微脉冲半导体激光对兔视网膜色素上皮细胞阈值下光凝的光生物调制效应

Photobiological effects of micropulsed subthreshold 810 nm diode laser photocoagulation on retinal pigment epithelial cells of rabbits

目的探讨微脉冲激光阈值下光凝对色素兔视网膜形态结构以及视网膜色素上皮细胞(retinal pigment epithelial cells,RPE)分泌细胞因子的影响。方法微脉冲810 nm激光阈值下光凝色素兔视网膜,光凝后1 d、3 d、7 d、14 d分别行眼底荧光血管造影(fluorescein angiography,FFA)和组织切片技术观察视网膜结构变化;免疫荧光技术观察视网膜血管内皮生长因子(vascular endothelial growth factor,VEGF)、色素上皮源性因子(pigment epithelium-derived factor,PEDF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,b-FGF)的表达变化;RT-PCR检测视网膜组织匀浆中VEGF mRNA、PEDF mRNA、b-FGF mRNA的表达变化。结果光凝后FFA检查未见荧光素渗漏;组织切片各层结构基本完整。免疫荧光表明,光凝后VEGF、PEDF在视细胞层及RPE层表达明显增强;b-FGF在神经纤维层、节细胞层也有表达。RT-PCR表明,激光后3种细胞因子mRNA含量均增多,光凝后1 d PEDF mRNA(3.748±0.890)表达最高,3 d次之,与其他时间点比较差异均有统计学意义(均为P<0.05);b-FGF mRNA光凝后1 d(1.578±0.299)增高最明显,但与其他时间点比较差异均无统计学意义(均为P>0.05);光凝后3 d VEGF mRNA(2.301±0.378)表达最高,与其他时间点比较差异均有统计学意义(均为P<0.05)。光凝后14 d,3种细胞因子的升高幅度相近(1.283±0.310、1.662±0.409、1.310±0.184)。结论微脉冲810 nm激光阈值下光凝可刺激正常视网膜RPE细胞协调表达分泌3种细胞因子,且对视网膜组织结构无损伤。

Objective To investigate the photobiological effects of micropulsed subthreshold 810 nm diode laser photocoagulation on retinal structure and cell factor se cretion under retinal pigment epithelial cells of chinchilla rabbits. Methods Micro pulsed subthreshold 810 nm diode laser photocoagulated retina of chinchilla rabbits. By fluorescein angiography （FFA）, HE staining of retina, the changes of morphology and vascular of retina were observed at 1 day,3 days,7 days, 14 days after photocoagula tion. The expression changes of vascular endothelial growth factor （VEGF） ,pigment ep ithelium-derived factor （PEDF） ,basic flbroblast growth factor （b-FGF） in retina were observed by immunofluorescence. The mRNA expression changes of VEGF, PEDF, b- FGF were analyzed in retinal tissue homogenates by RT-PCR. Results After photoco- agulation, FFA examination showed no vascular fluorescence leakage;Retinal tissue sec tions stained with HE showed retina structure was in order generally;Immunofluores cence showed that after laser photocoagulation, the expression of VEGF and PEDF were enhanced significantly in rods or cones cell layer and retinal pigment epithelial layer ;b FGF was also expressed in nerve fiber layer and ganglion cell layer. RT-PCR results showed that after laser photocoagulation the mRNA content of three cytokines in creased. PEDF mRNA expression increased to the maximum at I day after photocoagu lation （ 3. 748 ± 0. 890）, followed by that at 3 days, which both had significantly differ ence compared with the other time points （ all P 〈 0.05 ）. b-FGF mRNA expression in creased to the maximum at 1 day after photocoagulation （ I. 578 ± 0. 299 ）, but for each time point, the difference was not statistically significant（ all P 〉 0.05 ）. VEGF mRNA ex- pression increased to the maximum at 3 days after photocoagulation （ 2. 301 ± 0. 378 ）, which had significantly difference compared with other time points（ all P 〈0.05）. At 14days after photocoagulation,the increased amplitude of three cytokines was near to each other （1. 283 ± 0. 310,1. 662± 0. 409,1.3 l0 ± 0. 184 ）. Conclusion Micropulsed subthreshold 810 nm diode laser can stimulate normal RPE cells in vivo to secrete VEGF,PEDF,b-FGF in perfect union with no damage on retinal structure.