Abels C, Fickweiler S, Weiderer P, Bäumler W, Hofstädter F, Landthaler M, Szeimies R M
Department of Dermatology, University of Regensburg, Germany.
Arch Dermatol Res. 2000 Aug;292(8):404-11. doi: 10.1007/s004030000147.
The cellular uptake and subcellular localization of indocyanine green (ICG; absorption band 700-850 nm), and cell survival and ultrastructural changes following ICG-mediated phototherapy were investigated in vitro in four different cell lines derived from human skin (SCL1 and SCL2 squamous cell carcinoma, HaCaT keratinocytes and N1 fibroblasts). The cellular uptake of ICG (1-50 microM, incubation times 1, 4, 24 h) was saturable, highly cumulative and could be inhibited by the addition of 250 microM bromosulphophthalein indicating the involvement of the organic anion transporting polypeptide (OATP). For HaCaT cells, the maximum cellular uptake (Vmax) and the Michaelis constant (K(m)) were 9.9 +/- 1.1 mM and 47 +/- 16 microM, respectively, following a 24-h incubation with ICG. Fluorescence microscopy revealed a cytoplasmic distribution of ICG, probably bound to glutathione S-transferase. Following irradiation with a cw-diode laser (805 nm, 80 mW/cm2) at doses of 24 or 48 J/cm2, the phototoxicity was determined using the MTT assay as a measure of cell viability. For all cell lines, ICG concentrations above 25 microM produced a significant phototoxic effect. The EC50, of ICG for HaCaT cells following irradiation at 24 J/cm2 was 20.1 +/- 3.9 microM. Growth curves showed that even HaCaT cells treated at the EC50 were killed within a week following treatment. Electron microscopy 1 h after ICG-mediated phototherapy revealed cytoplasmic vesiculation, dilation of the rough endoplasmic reticulum, the Golgi complex and the perinuclear cisternae and the beginning of chromatin condensation in the nucleus. These ultrastructural findings are not consistent with a photothermal action of ICG-mediated phototherapy. Taken together with those of previous studies by our group these results support photooxidation as a major cell-killing mechanism.
研究了吲哚菁绿（ICG；吸收带700 - 850 nm）在源自人皮肤的四种不同细胞系（SCL1和SCL2鳞状细胞癌、HaCaT角质形成细胞和N1成纤维细胞）中的细胞摄取和亚细胞定位，以及ICG介导的光疗后的细胞存活和超微结构变化。ICG（1 - 50 microM，孵育时间1、4、24小时）的细胞摄取是可饱和的、高度累积的，并且加入250 microM溴磺酞可抑制其摄取，这表明有机阴离子转运多肽（OATP）参与其中。对于HaCaT细胞，与ICG孵育24小时后，最大细胞摄取量（Vmax）和米氏常数（K(m)）分别为9.9 +/- 1.1 mM和47 +/- 16 microM。荧光显微镜显示ICG呈细胞质分布，可能与谷胱甘肽S - 转移酶结合。用连续波二极管激光（805 nm，80 mW/cm2）以24或48 J/cm2的剂量照射后，使用MTT法测定光毒性以衡量细胞活力。对于所有细胞系，ICG浓度高于25 microM产生显著的光毒性作用。在24 J/cm2照射后，HaCaT细胞的ICG半数有效浓度（EC50）为20.1 +/- 3.9 microM。生长曲线表明，即使是在EC50浓度下处理的HaCaT细胞在处理后一周内也会死亡。ICG介导的光疗1小时后的电子显微镜检查显示细胞质空泡化、粗面内质网、高尔基体复合体和核周池扩张以及细胞核中染色质开始凝聚。这些超微结构发现与ICG介导的光疗的光热作用不一致。结合我们小组之前的研究结果，这些结果支持光氧化作为主要的细胞杀伤机制。
