Experimental Research on Subcellular Distribution of PpIX Photobleaching in HL60 Based on ALA-PDT
随着光动力学疗法(photodynamic therapy，PDT)基础研究的不断深入和临床应用的广泛开展，光敏剂的光漂白现象日渐成为PDT研究的热点问题。实验在ALA的终浓度为10 mM/ml，辐射光波长为410 nm，辐照光功率为5 mW/cm2等条件下采用荧光探针标记技术在自行设计的光动力疗法反应室进行了PDT实验研究。Cell Counting Kit-8 (CCK-8)法用来检测细胞的活性，通过获得的荧光光谱研究了亚细胞的光漂白情况。结果表明：以每种细胞器光照前的平均荧光强度为基准，则光照60 min后线粒体、溶酶体、内质网的荧光强度下分别降低了95%、91.5%和51.2%。线粒体区域光漂白速率较快，其荧光强度的变化基本表征了HL60细胞活性的变化，推断线粒体是光动力效应的主要作用靶点之一。
Abstract: With the development of the fundamental research of photodynamic therapy (PDT) and its clinical applica-tion, the photobleaching properties of photosensitizers have become a hotspot of research. The experimental research of ALA mediated PDT was carried out in the own designed reaction chamber when the final concentration of ALA is 10 mM/ml, the wavelength and the output power of the light source are 410 nm and 5 mW/cm2 separately. Cell Counting Kit-8 (CCK-8) was used to evaluate the cell viability while the photobleaching of PpIX in the subcellular structure of HL60 was detected by fluorescence spectroscopy. The results showed that after 60 min irradiation the fluorescence in-tensity of mitochondria, lysosomes and endoplasmic reticulum had decreased by 95%, 91.5% and 51.2% respectively compared to that before irradiation. The rate of photobleaching in mitochondria which can characterize the activity of HL60 was significantly higher than others, this suggest that mitochondria might be one of main therapeutic targets of photodynamic therapy.
文章引用: 黄康强 , 吕顺峰 , 熊建文 (2012) 基于ALA-PDT中PpIX亚细胞分布位点光漂白实验研究。 现代物理， 2， 94-100. doi: 10.12677/MP.2012.24016
 J. S. Dysart, M. S. Patterson, T. J. Farrell, et a1. Relationship between mTHPC fluorescence photobleaching and cell viability during in vitro photodynamic treatment of DP16 cells. Photochemistry and Photo-biology, 2002, 75(2): 289-295.
 J. Tyrrell, S. M. Campbell and A. Curnow. Monitoring the accumulation and dissipation of the photosen-sitizer protoporphyrin IX during standard dermatological methyl-aminolevulinate photo- dynamic therapy utilizing noninvasive fluorescence imaging and quantification. Photodiagnosis and Photo-dynamic Therapy, 2011, 8(1): 30-38.
 C. P. Chang, D. J. Nagel and M. E. Zaghloul. Irradiance dependence of photobleaching of resoruﬁn. Journal of Photochemistry and Photobiology A: Chemistry, 2011, 217(2): 430-432.
 J. S. Dysart, G. Singh and M. S. Patterson. Calculation of singlet oxygen dose from photosensitizer fluorescence and photobleaching during mTHPC photodynamic therapy of MLL cells. Photochemistry and Photobiology, 2005, 81(1): 196–205.
 J. S. Dysart, M. S. Patterson. Characterization of photofrin photo- bleach-ing for singlet oxygen dose estimation buring photodynamic therapy of MLL cells in vitro. Physics in Medicine and Biology, 2005, 50(11): 2597-2616.
 W. S. Strauss, R. Sailer, M. H. Gschwend, et a1. Selective examination of plasma membrane associated photosensitizers Using total internal reﬂection fuorescence spectroscopy: Correlation between photobleaching and photodynamic efﬁcacy of protoporphyrin IX. Photochemistry and Photobiology, 1998, 67(3): 363-369.
 P. M. Msc, A. Juzenierea and J. Moan. The effect of lidocaine on PpIX photobleaching and outcome of ALA-PDT in vitro. Photodiagnosis and Photodynamic Therapy, 2007, 4(4): 249-253.
 I. A. Boere, D. J. Robinson, H. W. Tilanus, et a1. Monitoring in situ dosimetry and pro-toporphyrin IX fuorescence photobleaching in the normal rat esopha-gus during 5-aminolevulinic acid photodynamic therapy. Photochemis-try and Photobiology, 2003, 78(3): 271-277.
 H. Zeng, M. Korbelik, D. I. Mclean, et a1. Monitoring photoproduct formation and photobleaching by fuorescence spectroscopy has the potential to improve PDT dosimetry with a verte- porﬁn-like photosensitizer. Photochemistry and Photobiology, 2002, 75(4): 398-405.
 J. Moan, G. Streckyte, S. Bagdonas, et a1. Photobleaching of protoporphyrin IX in cells incu-bated with 5-aminolevulinic acid. International Journal of Cancer, 1997, 70(1): 90-97.
 J. Tyrrell, S. M. Campbell and A. Curnow. Pro-toporphyrin IX photobleaching during the light irradiation phase of standard dermatological methyl-aminolevulinate photodynamic therapy. Photodiagnosis and Photodynamic Therapy, 2010, 7(4): 232-238.
 熊建文, 肖化, 张镇西. MTT法和CCK-8法检测细胞活性之测试条件比较[J]. 激光生物学报, 2007, 16(5): 526-531.
 张镇西, 张苏娟, 张宝琴等. 氨基酮戊酸光动力学疗法杀伤白血病细胞的实验研究[J]. 中国激光医学杂志, 2005, 14(4): 249-252.
 肖化, 熊建文, 吴继明等. ALA-PDT对白血病肿瘤细胞作用的参数研究[J]. 激光生物学报, 2004, 13(5): 353-357.
 M. B. Ericson, F. Gudmundson, A. M. Wennberg, et al. A spectroscopic study of the photobleaching of protoporphyrin IX in solution. Lasers in Medical Science, 2004, 18(1): 56-62.
 J. Tyrrell, S. M. Campbell and A. Curnow. The effect of air cooling pain relief on protoporphyrin IX photobleaching and clini-cal efﬁcacy during bermatological photodynamic therapy. Journal of Photochemistry and Photobiology B: Biology, 2011, 103(1): 1-7.
 J. Moan and K. Berg. The photodegradation of porphyrins in cells can be used to estimate the lifetime of snglet oxygen. Pho- to-chemistry and Photobiology, 1991, 53 (4): 549-553.
 S. Bagdonas, L. W. Ma, P. Juzenas, et al. Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: A spectroscopic study. Photo-chemistry and Photobiology, 2000, 72(2): 186-192.