组织因子靶向肽对蓝光诱导的人视网膜色素上皮细胞损伤的保护作用

Protective effects of tissue factor targeting peptide on human retinal pigment epithelial cell damage induced by blue light

背景视网膜光损伤可造成视网膜色素上皮（RPE）细胞损伤，是影响年龄相关性黄斑变性（AMD）发生和发展的重要因素之一。研究表明，组织因子（TF）在氧化损伤的RPE细胞中和AMD患者的脉络膜新生血管（CNV）中呈高表达，推测抑制TF可预防RPE细胞的损伤以及抑制CNV。目的观察TF靶向肽（TF-TP）对蓝光诱导的人RPE细胞损伤的保护作用。方法体外分离和培养人RPE细胞并分为空白对照组、蓝光照射组和TF-TP组。空白对照组细胞在常规条件下进行处理；蓝光照射组细胞用辐照强度为（4.0±0.5）mW/cm2的蓝光照射12 h建立蓝光损伤细胞模型；TF-TP组先分别用不同浓度（10、100、150、200、300 μmol/L）TF-TP培养细胞24 h，再用蓝光照射12 h。采用CCK-8法检测各组细胞的存活率；分别在普通倒置显微镜和透射电子显微镜下观察RPE细胞的形态和超微结构变化；采用Hoechst染色法检测各组细胞的凋亡情况；采用Western blot法检测各组细胞中TF蛋白和相关凋亡蛋白bax和bcl-2的表达。结果不同浓度TF-TP组间RPE细胞存活率比较，差异无统计学意义（F＝2.15，P＝0.11）。空白对照组、蓝光照射组和150 μmol/L TF-TP组细胞存活率分别为（100.0±0.00）%、（43.79±6.55）%和（63.45±3.57）%，150 μmol/L TF-TP组细胞存活率较蓝光照射组明显增加，差异有统计学意义（P＝0.00），以150 μmol/L TF-TP为后续实验的最适浓度。光学显微镜下和透射电子显微镜下显示，蓝光照射组有较多皱缩、变形、悬浮细胞，可见细胞微绒毛数量减少，部分线粒体嵴断裂和缺失以及细胞空泡样变性，而150 μmol/L TF-TP组异常形态的细胞较少，细胞绒毛结构较完整，细胞质中空泡样结构改变和线粒体损伤改变明显减轻。空白对照组、蓝光照射组和150 μmol/L TF-TP组细胞凋亡率分别为（0.98±0.19）%、（9.98±0.82）%和（5.73±0.88）%，组间总体比较差异有统计学差异（F＝206.18，P＝0.00），其中150 μmol/L TF-TP组细胞凋亡率明显低于蓝光照射组，差异有统计学意义（P〈0.05）。与空白对照组相比，蓝光照射组细胞中bax蛋白和TF蛋白的相对表达量明显增加，bcl-2蛋白的相对表达量显著下降，差异均有统计学意义（均P〈0.05）；与蓝光照射组相比，150 μmol/L TF-TP组细胞中bax蛋白和TF蛋白的相对表达量均明显下降，而bcl-2蛋白的相对表达量明显增加，差异均有统计学意义（均P〈0.05）。结论TF-TP预处理后可减少蓝光损伤的人RPE细胞的凋亡及增加细胞的存活率，从而对蓝光诱导的人RPE细胞损伤发挥保护作用，其作用机制可能与TF-TP抑制TF介导的bax/bcl-2凋亡通路有关。

BackgroundLight-induced retinal damage results in the damage of retinl pigment epithelial （RPE） cells and therefore affects the pathogenesis and development of age-related macular degeneration （AMD）. Studies showed that tissue factor （TF） is overexpressed in oxidative damaged RPE cells and the choroidal neovascularization （CNV） of AMD, speculating that the suppression of TF can prevent the damage of RPE cells and inhibit CNV.ObjectiveThis study was conducted to observe the protective effects of TF targeting peptide （TF-TP）, a new drug of autologous synthesis, on human RPE-cells induced by blue light.MethodsHuman RPE cells were isolated from donor eye and cultured.Cultured cells were divided into blank control group, model group and TF-TP treated group.Light-induced RPE cell damage model was established by exposuring the cells in the blue light of （4.0±0.5）mW/cm2 for 12 hours in the model group, and different concentrations （10, 100, 150, 200, 300 μmol/L） of TF-TP were added into the medium to pretreat the cells for 24 hours and then exposed the cells to the blue light for 12 hours in the TF-TP groups.The cell viability was determined by CCK-8 assay.The morphology and ultrastructure in the cells were observed under the inverted microscope and transmission electron microscope.The apoptosis of the cells was assayed by Hoechst staining.The expressions of TF and apoptosis-related protein bax, bcl-2 in the cells were determined by Western blot.ResultsCCK-8 assay showed that there was no significant difference in the cell viability among blank control group and different concentrations TF-TP groups （F=2.15, P=0.11）. The cell survival rate of blank control group, model group and 150 μmol/L TF-TP group was （100.0±0.00）%, （43.79±6.55）% and （63.45±3.57）%, and the survial rate was increased in the 150 μmol/L TF-TP group compared with the model group （P=0.00）, and 150 μmol/L was detemined as a optimal concentration of TF-TP.A lot of shrinkage, deformation, suspension cells were exhibited under the optical microscope, and decrease of microvilli structure, rupture of mitochondrial cristae and vacuolar degeneration of the cells were found in the model group, and the damage of the cells were evidently lightened in the 150 μmol/L TF-TP group.The apoptosis rate of the cells were （0.98±0.19）%, （9.98±0.82）% and （5.73±0.88）% in the blank group, model group and 150 μmol/L TF-TP group, respectively, with a significant difference among the groups （F=206.18, P=0.00）, and the apoptosis rate of the cells in the 150 μmol/L TF-TP group was significantly lower than that in the model group （P〈0.05）. Compared with the blank control group, the relative expression of bax and TF was obviously increased and that of bcl-2 was decreased in the model group; while the expression of bax and TF was lower, and that of bcl-2 was higher in the 150 μmol/L TF-TP group compared with the model group （all at P〈0.05）.ConclusionsPretreation of TF-TP can lessen cell apoptosis and increase cell survival rate and therefore plays a protective role to blue light-induced human RPE cells possibly by inhibiting bax/bcl-2 apoptotic pathways mediated by TF.