目的 探讨水溶性半乳糖酞菁类光敏剂T1联合光动力疗法(PDT)对人肝癌细胞系的体外抑瘤效应和对小鼠肝癌H-22的体内抑瘤效果.方法 取对数生长期HepG2细胞培养,分设不同浓度T1给药组(0 μM、0.06 μM、0.125 μM、0.25 μM、0.5 μM、1μM).利用四甲基偶氮唑法检测PDT对HepG2增殖的影响,用激光共聚焦显微镜检测T1在HepG2细胞中的亚细胞定位,利用JC-1染色和高内涵细胞成像分析仪检测细胞凋亡及坏死情况,检测T1-PDT后HepG2细胞中活性氧(ROS)含量变化,利用RT-PCR检测Bcl-2、Bax mRNA的表达水平.选取健康小鼠24只,建立小鼠肝癌H-22荷瘤小鼠模型,按照随机数字表法将其分为4组,分别为对照组、低剂量组、中剂量组、高剂量组,每组4只,给予不同剂量T1-PDT,计算抑瘤率.结果 四甲基偶氮唑法结果表明一定剂量的单纯光敏剂或激光对肿瘤细胞的生长无影响或影响较小,而T1-PDT治疗能明显抑制HepG2肿瘤细胞的增殖,诱导HepG2细胞凋亡,T1存在于HepG2细胞的线粒体内,能促使HepG2内ROS含量增加,RT-PCR结果显示Bax的表达水平升高、Bcl-2表达水平降低.体内实验显示T1-PDT可以显著抑制小鼠移植瘤生长.结论 T1介导的PDT可以有效诱导HepG2细胞凋亡,其机制可能与T1的亚细胞定位、增加胞内ROS的含量、调节凋亡相关基因有关.%Objective To explore the therapeutic efficacy of the water soluable photosensitizer D-galactopyranosyl zinc phthalocyanines (T1)-mediated photodynamic therapy (PDT) applied to HepG2 human hepatocellular carcinoma cells in vitro and in vivo,as well as its mechanism.Methods HepG2 cells in their logarithmic growth phase were cultured and divided into different concentrations ofT1 (0 μM,0.06 μM,0.125 μM,0.25 μM,0.5 μM and 1 μM).Methyl thiazolyl tetrazolium (MTT) assays were employed to determine the effect of the T1-PDT on the proliferation of the HepG2 cells.Cell apoptosis and necrosis were measured using a cell analyzer with Annexin VFITC/PI/Hochest33342 triple-staining.The reactive oxygen species (ROS) and the mitochondrial membrane potentials of the HepG2 cells were detected using fluorescence microscopy.Confocal microscopic assays were used to observe T1's subcellular localization on the HepG2 cells.Real-time quantitative polymerase chain reactions (RT-PCRs)were used to detect any apoptosis of Bcl-2-and Bax-related genes.H-22-bearing mice were used to calculate the antitumor rate of T1-PDT,and the expression levels of Bcl-2 and Bax mRNA were detected using RT-PCRs.Twenty-four healthy mice were randomly divided into a control group,a low-dose group,a middle-dose group and a high-dose group,each of 6.Each group was given different doses of T1-PDT and the tumor inhibition rate was calculated.Results The MTT assays showed that T1-PDT could significantly inhibit HepG2 cell growth,but T1 or PDT alone had little effect.The confocal microscope assay showed that T1 was mainly localized in the mitochondria in HepG2 cells with little in the lysosome.Cell analyzer results showed that T1-PDT could induce HepG2 apoptosis.The ROS levels of HepG2 cells increased after T1-PDT.The RT-PCR results showed that T1-PDT could increase the expression of Bax and decrease the expression of Bcl-2.The in vivo experiments demonstrated that T1-PDT significantly inhibited the growth of H-22-xenografied tumors.Conclusions T1-mediated PDT has a significant lethal effect on HepG2 cells in vitro and in vitro.The lethal effect of PDT on cancer cells is shown in the apoptosis and can be attributed to T1's subcellular localization in the mitochondria,increasing ROS levels,and regulating apoptosis-related genes.
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