酞菁锌介导的光动力学疗法对骨髓净化的实验研究

目的 研究酞菁锌光敏剂(ZnPcS2P2)介导的光动力学疗法(PDT)对模拟慢性粒细胞白血病缓解骨髓的净化作用。方法 采用荧光分光光度法检测K562细胞及正常细胞内的光敏剂含量。锥虫蓝拒染法、MTT比色法、克隆形成实验检测不同浓度ZnPcS2P2介导的PDT对K562细胞增殖能力的影响。正常混合集落形成细胞(CFU-Mix)、粒一单核系造血祖细胞(CFU-GM)、红系祖细胞(CFu-E)等集落形成实验检测ZnPcS2P2 PDT对正常造血祖细胞的影响。巢式PCR方法检测酞菁锌介导的：PDT作用前后混有不同比例K562细胞的骨髓细胞的bcr-abl mRNA表达。结果 (1)与ZnPcS2P2共孵育5h,K562细胞与骨髓正常单个核细胞(MNC)内的ZnPcS2P2含量分别为10．1、2．2(ng／5x10^5细胞),二者比值达到最高值。(2)0．25μg／ml ZnPcS2P2孵育5h后,用670nm激光以53mW／cm^2的功率密度、2．1J／cm^2的能量密度照射对K562细胞集落形成的抑制率为91．1％,而对正常CFU-Mix、CFU-GM、CFU-E等集落形成的抑制率分别为18．0％、18．6％、17．8％。(3)0．25μg／ml ZnPcS2P2介导的PDT可完全杀灭按1：100至1：1000比例混入骨髓正常MNC中的K562细胞。结论 ZnPcS2P2介导的PDT能选择性杀伤K562细胞,有希望成为新的高效而简便的慢性粒细胞白血病骨髓净化手段。

Effect of zinc phthalocyanine-mediated photodynamic therapy on bone marrow purging,an experimental study

OBJECTIVE: To probe into the purging effects of zinc phthalocyanine-mediated photodynamic therapy (PDT) on simulated remission bone marrow grafts of chronic granulocytic leukemia. METHODS: (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. RESULTS: (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. CONCLUSION: Zinc phthalocyanine -based PDT selectively kills K562 cells. It would be a promising purging technique for chronic granulocytic leukemia.