光敏剂间-四羟基苯二氢卟酚诱导肿瘤细胞凋亡的研究

Apoptosis Induction in Tumor Cells by Photosensitizer Mesotetrahydroxyphenylchlorin

目的：探讨体外以间－四羟基苯二氢卟酚 (mesotetrahydroxyphenylchlorin,mTHPC)为光敏剂的光动力诱导肿瘤细胞死亡的机制。方法：用含 1.3μ mol/L mTHPC (0.8μ g/ml)的培养液孵育人骨肉瘤细胞系 (HOS-8603)和小鼠髓性白血病细胞系 (命名为 JCS细胞 )细胞 16 h后用波长 580 nm、 7.5 mW/cm2光照射。观察光处理后瘤细胞存活率的变化，用共聚焦显微镜检测 mTHPC在瘤细胞的分布；用 Hoechst 33258荧光染色显示瘤细胞的凋亡形态变化，而瘤细胞凋亡 DNA片段用琼脂糖电泳检测。结果： mTHPC位于 HOS-8603和 JCS细胞的胞浆，经光动力治疗后，细胞存活率随培养时间延长逐渐降低， JCS细胞半数存活率为 2 h， HOS-8603细胞为 4 h。 JCS细胞凋亡率在处理后 1 h为 2.1％、 2 h为 62.9％、 4 h为 100.0％，而 HOS－ 8603细胞凋亡率 4 h为 20.2％、 8 h为 65.9％、 12 h为 100.0％。两种细胞在电泳中均出现特征性凋亡条带。结论： mTHPC是一种强光效应的光敏剂，能有效地进入肿瘤细胞胞浆，诱导瘤细胞死亡的方式主要是凋亡，其次是坏死。

Objective： This study was designed to investigate the death mechanism of tumor cells after photodynamic therapy with mesotetrahydroxyphenylchlorin(mTHPC) as a photosensitizer. Methods： Human osteosarcoma cells (HOS-8603) and murine myeloid leukemia cells(JCS) were treated with 1.3μ mol/L mTHPC for 16 hours in vitro, and then exposed to 7.5 mW/cm2 intensity of 580 nm light. Cell survival was calculated and intracellular localization of mTHPC was observed by confocal laser scanninig microscopy. Apoptotic morphology of the cells was demonstrated by fluorescence staining of Hoechst 33258. Apoptotic DNA ladders were detected by agarose electrophoresis. Results： Fluorescence of mTHPC was observed in the cytoplasm of two cell types. After photodynamic therapy, cell survival rate was decreased with prolongation of incubation time. Fifty percent of cell killing was at 2 hours in JCS cells, and at 4 hours in HOS-8603 cells after light treatment. The percentages of apoptotic cells in JCS were 2.1％ at 1 h, 62.9％ at 2 h and 100％ at 4 h, and in HOS-8603 were 20.2％ at 4 h, 65.9％ at 8 h and 100％ at 12 h, respectively. A characteristic apoptotic ladders were showed in all cell types. Conclusion： The mTHPC is a potent photosensitizer and can penetrate into cytoplasm of the cells effectively. Induced cell death of photodynamic therapy is mainly apoptosis followed by necrosis.