Huang Hui-fang, Chen Yuan-zhong, Wu Yong
Fujian Institute of Hematology, Union Hospital, Fujian Medical University, Fuzhou 350001, China.
Zhonghua Yi Xue Za Zhi. 2003 Jun 10;83(11):986-91.
To probe into the purging effects of zinc phthalocyanine-mediated photodynamic therapy (PDT) on simulated remission bone marrow grafts of chronic granulocytic leukemia.
(1) K562 cells, aline chronic granulocytic leukemia cells, and normal mononuclear cells (MNC) were cultured. Zinc phthalocyanine (ZnPcS(2)P(2)), a photosensitizer, with the terminal concentration of 1.0 micro g/ml was added into the cultures. The K562 cells and normal MNCs in the suspensions were broken. Fluorescence spectrophotometry was used to determine the concentration of zinc phthalocyanine in cells at different time points so as to find the optimal time for photodynamic purging process. (2) Suspensions of K562 cells and MNCs were made and incubated with zinc phthalocyanine of different concentrations (0.062 5, 0.125, 0.25, 0.5, and 1.0 micro g/ml) for 5 hours. A blank control group (sodium chloride of the same volume was added), a PDT control group (without photosensitizer), and a photosensitizer control group (zinc phthalocyanine was added without PDT) were established. Then the suspensions were irradiated with 670 nm laser. Trypan blue dye exclusion technique was used to calculate the number of live cells for a period of 5 days. The proliferative potency of K562 cells was detected by MTT colorimetric assay. The OD value was detected with ELISA apparatus to calculate the inhibition rate. Colony formation of K562 cells and MNCs was determined. (3) K562 cells were mixed into normal MNCs at the ratios of 1:100 and 1:1,000 so as to create the model of simulated remission bone marrow. After PDT treatment, colony formation test was done and nested-PCR was used to detect the bcr-abl mRNA expression in K562 cells. Colony formation test was made on the MNCs treated with PDT. The antiproliferative effects of PDT on normal hematopoietic progenitors were evaluated by CFU-Mix, CFU-GM and CFU-E assays.
(1) The zinc phthalocyanine content in the MNCs reached its peak within the first hour of incubation and then rapidly decreased to the lowest value in 4 hours. However, the zinc phthalocyanine content in the K562 cells gradually increased within the first 4 hours of incubation and reached its peak by the fifth hour with a ratio of zinc phthalocyanine content in K562 cells to that in MNCs of 4.59. Therefore, the fifth hour after incubation was selected as the optimal time to irradiate the suspensions using the laser with a wavelength of 670 nm. (2) The inhibitory rate of laser on the colony information rate was 91.1% for the K562 cells, 18.0% for the MNCs in CFU-Mix methyl cellulose culture system, 18.6% for the MNCs in CFU-GM methyl cellulose culture system, and 17.8% for the MNCs in CFU-E methyl cellulose culture system At the concentration of 0.25 micro g/ml, K562 cells were inhibited by 91.1%, however, CFU-Mix, CFU-GM and CFU-E were relatively spared, inhibitory rate being 18.0%, 18.6% and 17.8% respectively. (3) At the concentration of 0.25 micro g/ml, residual K562 cells in the simulated remission bone marrow were completely photoinactivated.
Zinc phthalocyanine -based PDT selectively kills K562 cells. It would be a promising purging technique for chronic granulocytic leukemia.
探讨锌酞菁介导的光动力疗法（PDT）对慢性粒细胞白血病模拟缓解期骨髓移植物的清除效果。
（1）培养K562细胞、慢性粒细胞白血病细胞系及正常单个核细胞（MNC）。向培养物中加入终浓度为1.0μg/ml的光敏剂锌酞菁（ZnPcS(2)P(2)）。将悬液中的K562细胞和正常MNC破碎。采用荧光分光光度法测定不同时间点细胞内锌酞菁的浓度，以找出光动力清除过程的最佳时间。（2）制备K562细胞和MNC悬液，与不同浓度（0.062 5、0.125、0.25、0.5和1.0μg/ml）的锌酞菁孵育5小时。设立空白对照组（加入等体积氯化钠）、PDT对照组（不加入光敏剂）和光敏剂对照组（加入锌酞菁但不进行PDT）。然后用670nm激光照射悬液。采用台盼蓝拒染法计算5天内活细胞数量。采用MTT比色法检测K562细胞的增殖能力。用酶标仪检测OD值并计算抑制率。检测K562细胞和MNC的集落形成情况。（3）将K562细胞按1:100和1:1 000的比例混入正常MNC中，构建模拟缓解期骨髓模型。PDT治疗后，进行集落形成试验，并用巢式PCR检测K562细胞中bcr-abl mRNA的表达。对经PDT处理的MNC进行集落形成试验。通过CFU-Mix、CFU-GM和CFU-E测定评估PDT对正常造血祖细胞的抗增殖作用。
（1）孵育第1小时内MNC中锌酞菁含量达到峰值，然后在4小时内迅速降至最低值。然而，K562细胞内锌酞菁含量在孵育的前4小时逐渐增加，第5小时达到峰值，K562细胞与MNC中锌酞菁含量之比为4.59。因此，选择孵育后第5小时作为用670nm激光照射悬液的最佳时间。（2）在CFU-Mix甲基纤维素培养体系中，激光对K562细胞集落形成率的抑制率为91.1%，对MNC的抑制率为18.0%；在CFU-GM甲基纤维素培养体系中，对MNC的抑制率为18.6%；在CFU-E甲基纤维素培养体系中，对MNC的抑制率为17.8%。在0.25μg/ml浓度时，K562细胞被抑制91.1%，而CFU-Mix、CFU-GM和CFU-E相对受影响较小，抑制率分别为18.0%、18.6%和17.8%。（3）在0.25μg/ml浓度时，模拟缓解期骨髓中残留的K562细胞被完全光灭活。
基于锌酞菁的PDT可选择性杀伤K562细胞。它可能是一种有前景的慢性粒细胞白血病清除技术。
