Huang Yan, Xu Guoxing, Peng Yiru, Chen Shaoqiang, Wu Yunxia
Department of Ophthalmology and Optometry, Fujian Medical University, Fuzhou, Fujian, China.
J Ocul Pharmacol Ther. 2009 Jun;25(3):231-8. doi: 10.1089/jop.2008.0058.
The objective of this study was to investigate the effects of phototoxicity on retinal pigment epithelial (RPE) cells after zinc tetrasulfonated phthalocyanine bound bovine serum albumin (ZnPcS(4)-BSA) based photodynamic therapy (PDT). This study will provide a rational basis for the development of new clinical perspectives in future choroidal neovascularization (CNV) therapy.
The 2-4 generation of subcultured RPE cells were used in this study. First, the cellular uptake of ZnPcS(4) and ZnPcS(4)-BSA was quantified by UV-spectra after confluent human RPE cells were exposed to various doses of ZnPcS(4) and ZnPcS(4)-BSA for 1-6 h. Second, RPE cells were divided into the following five groups: the control group, the group of photosensitizer without irradiation, the irradiated group without photosensitizer, PDT1 group (irradiated with 50 J/cm(2)), and the PDT2 group (irradiated with 100 J/cm(2)) that was exposed to 10 mg/mL ZnPcS(4)-BSA before they were irradiated. After ZnPcS(4)-BSA based PDT was performed, cell viability was evaluated by MTT, and reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsi(m)) were assessed by flow cytometry and confocal microscopy, respectively.
The ratios of ZnPcS(4)-BSA content in the RPE cells were more than the intake ratios of ZnPcS(4) incubation at the same time point. It was also found that the optimal time point to irradiating the cell was 4 h after incubation. The cellular viability reduction ratios in the PDT1 group and PDT2 group RPE cell after ZnPcS(4)-BSA based PDT were 60.71% and 90.48%, respectively, which were higher than that of ZnPcS(4)-based PDT. PDT results in a significant increase in ROS. The mitochondrial membrane potentials of PDT1 group and PDT2 group were significantly decreased compared with the normal control group.
The ratio of ZnPcS(4)-BSA content in RPE cells reached its peak after 4 h incubation. Intracellular ROS formation and the loss of Deltapsi(m) may play an important role in mediating the apoptotic processes of RPE cells induced by ZnPcS(4)-BSA based PDT. This study revealed that there is an important precondition to decrease the damage on RPE, that is, we should avoid irradiating RPE cells after the peak incubation time period.
本研究的目的是调查基于四磺化酞菁锌结合牛血清白蛋白（ZnPcS(4)-BSA）的光动力疗法（PDT）对视网膜色素上皮（RPE）细胞的光毒性作用。本研究将为未来脉络膜新生血管（CNV）治疗新临床观点的发展提供合理依据。
本研究使用第2 - 4代传代培养的RPE细胞。首先，将汇合的人RPE细胞暴露于不同剂量的ZnPcS(4)和ZnPcS(4)-BSA 1 - 6小时后，通过紫外光谱对ZnPcS(4)和ZnPcS(4)-BSA的细胞摄取量进行定量。其次，将RPE细胞分为以下五组：对照组、未照射的光敏剂组、未使用光敏剂的照射组、PDT1组（照射剂量为50 J/cm(2)）和PDT2组（照射剂量为100 J/cm(2)），PDT2组在照射前暴露于10 mg/mL ZnPcS(4)-BSA。基于ZnPcS(4)-BSA的PDT治疗后，通过MTT评估细胞活力，分别通过流式细胞术和共聚焦显微镜评估活性氧（ROS）和线粒体膜电位（ΔΨm）。
在同一时间点，RPE细胞中ZnPcS(4)-BSA含量与ZnPcS(4)孵育的摄取率相比更高。还发现照射细胞的最佳时间点是孵育后4小时。基于ZnPcS(4)-BSA的PDT后，PDT1组和PDT2组RPE细胞的细胞活力降低率分别为60.71%和90.48%，高于基于ZnPcS(4)的PDT。PDT导致ROS显著增加。与正常对照组相比，PDT1组和PDT2组线粒体膜电位显著降低。
RPE细胞中ZnPcS(4)-BSA含量在孵育4小时后达到峰值。细胞内ROS的形成和ΔΨm的丧失可能在介导基于ZnPcS(4)-BSA的PDT诱导的RPE细胞凋亡过程中起重要作用。本研究表明，减少对RPE损伤的一个重要前提是，应避免在孵育高峰期后照射RPE细胞。
