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Basic Research of Photodynamic Therapy on Vascular Endothelial Cells.

血管内皮细胞光动力治疗的基础研究。

基本信息

批准号：
13671849
负责人：
OBANA Akira
金额：
$ 1.34万
依托单位：
Osaka City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Phototoxicity of photodynamic therapy (PDT) using ATX-S10(Na) (ATX) and Verteporfin (BPD) was evaluated by MTS Assay on vascular endothelial cells and retinal pigment epithelial cells. PDT was conducted under two kinds of treatment conditions, short dye exposure PDT in which the cells were exposed to each photosensitizer for 5 minutes followed by laser irradiation without washing out the photosensitizer in the medium, and long dye exposure PDT, in which the cells were exposed to photosensitizers for more than 60 minutes, washed with phosphate-buffered saline, followed by laser irradiation using fresh medium. A diode laser of 670 nm wavelength was used for ATX-S10(Na) excitation and that of 689 nm for BPD.The degree of phototoxicity depended on the dye concentration and radiant exposure. In short-exposure PDT on human microvascular endothelial cells (CryoHMVEC-Ad) with a laser dose of 50 J/cm^2, the ED_<90> was 6.3 μg/ml of ATX-S10(Na) and 0.04 μg/ml of BPD, while in long-exposure PDT t … More he ED_<90> was 50.0 μg/ml of ATX and 0.04 μg/ml of BPD. Laser and dye doses to obtain ED_<90> became higher when the medium was supplemented with higher concentration of serum. In short-exposure PDT, the ED_<90> of monkey choroid-retina vascular endothelial cells (CRL-1780) was 100 μg/ml of ATX-S10(Na) and 0.08 μg/ml of BPD and an radiance of 100 J/cm^2. In comparison of phototoxicity on human retinal pigment epithelial cells (ARPE-19) and vascular endothelial cells, phototoxicity on vascular endothelial cells was significantly higher than that on the retinal pigment epithelial cells with short-exposure PDT using ATX, however, long-exposure PDT with ATX and short- and long-exposure PDT with BPD failed to obtain higher phototoxicity on vascular endothelial cells than on the retinal pigment epithelial cells. The uptake aid subcellular localization of ATX and BPD was examined by measuring fluorescence of each dye. ATX was mainly localized in the lysosomes and BPD in cytoplasma diffusely.Therefore, BPD had a higher phototoxicity than ATX. Some reasons were speculated for high phototoxibly of BPD, such as different amount and speed of uptake of the photosensitizer into the cells. In short-exposure PDT with ATX, vascular endothelial cells were selectively injured without damage on the retinal pigment epithelial cells, in contrast, with other PDT conditions, it seemed difficult to destroy vascular endothelial cells without damage on the retinal pigment epithelial cells since some dye was taken up into the retinal pigment epithelial cells.The mode of cell death by PDT was observed by TUNEL staining and a laser scanning cytometer. Short-exposure PDT with ATX predominantly induced necrosis, while long-exposure PDT resulted in apoptosis. Therefore, the dye that located on or surrounding the cell membrane seems to predominantly induced necrosis of the target cell, while the dye that accumulated in lysosomes predominantly induced apoptosis.ATX-S10 had a phototoxic effect on human skin malignant melanoma cells. Most of the dead cells appeared apoptotic with weak treatment conditions that induced low cytotoxicity. In contrast, most of them appeared necrotic with treatment conditions that induced more than 90% cytotoxicity. Less

采用MTS法检测ATX-S10（Na）（ATX）和维替泊芬（BPD）联合光动力疗法（PDT）对血管内皮细胞和视网膜色素上皮细胞的光毒性。PDT在两种处理条件下进行，短染料暴露PDT，其中细胞暴露于每种光敏剂5分钟，随后激光照射而不洗出培养基中的光敏剂，和长染料暴露PDT，其中细胞暴露于光敏剂60分钟以上，用磷酸盐缓冲盐水洗涤，随后使用新鲜培养基激光照射。ATX-S_（10）（Na）用670 nm半导体激光激发，BPD用689 nm半导体激光激发。在短时间照射人微血管内皮细胞（CryoHMVEC-Ad）的PDT中，激光剂量为50 J/cm ^2，<90>ATX-S10（Na）和BPD的艾德_分别为6.3 μg/ml和0.04 μg/ml，而在长时间照射PDT中，ATX-S10（Na）和BPD的ED分别为0.05 μg/ml和0.04 μg/ml。 ...更多信息 ATX的艾德_（ED）<90>为50.0 μg/ml，BPD为0.04 μg/ml。当<90>培养基中补充有较高浓度的血清时，获得艾德_的激光和染料剂量变高。在短时间曝光PDT中，<90>猴脉络膜-视网膜血管内皮细胞（CRL-1780）的艾德_为100 μg/ml ATX-S10（Na）和0.08 μg/ml BPD，辐射强度为100 J/cm ^2。在对人视网膜色素上皮细胞（ARPE-19）和血管内皮细胞的光毒性的比较中，使用ATX的短暴露PDT对血管内皮细胞的光毒性显著高于对视网膜色素上皮细胞的光毒性，然而，长时间暴露PDT与ATX和短-和长-暴露PDT与BPD未能获得更高的光毒性血管内皮细胞比视网膜色素上皮细胞。通过测量每种染料的荧光来检查ATX和BPD的摄取辅助亚细胞定位。ATX主要定位于溶酶体，而BPD弥散分布于胞浆，因此BPD的光毒性高于ATX。BPD的高光敏性可能与光敏剂进入细胞的量和速度不同有关。在短时间光动力疗法中，ATX可选择性地损伤血管内皮细胞而不损伤视网膜色素上皮细胞，而其他光动力疗法条件下，由于视网膜色素上皮细胞吸收了一些染料，因此很难在不损伤视网膜色素上皮细胞的情况下破坏血管内皮细胞。短时间暴露PDT与ATX主要诱导坏死，而长时间暴露PDT导致细胞凋亡。因此，位于细胞膜上或细胞膜周围的染料似乎主要诱导靶细胞的坏死，而聚集在溶酶体中的染料则主要诱导细胞凋亡。ATX-S10对人皮肤恶性黑色素瘤细胞具有光毒性作用。大多数死亡细胞在弱处理条件下出现凋亡，诱导低细胞毒性。相比之下，在诱导90%以上细胞毒性的处理条件下，大多数细胞出现坏死。少