Bystander cellular effects induced by low-fluence charged particles

我们研究了低通量低能碳离子束照射正常人成纤维细胞的旁观者细胞反应。通过在35 mm直径的组织培养皿的中心钻6.5mm的孔并在孔的底部附接2.5μ m厚的Mylar膜以提供用于低能碳离子束的窗口来构建辐照皿。将细胞接种到培养皿中的聚酯薄膜上，然后用6 MeV/n的碳离子以2 x 10^5粒子/cm ^2的能量密度进行辐照。该束流是由位于千叶的重离子医疗加速器（HIMAC）的中能束流课程产生的。使用4种不同的方案暴露细胞：（1）用Mylar膜上的所有细胞照射细胞;（2）用Mylar膜上的一半细胞群照射细胞;（3）以1：1的比例合并经照射的细胞和未照射的对照细胞，并作为单一培养物铺板;（4）照射一半细胞，并用间隙连接介导的细胞-细胞通讯的特异性抑制剂（40μM linodine）处理。用集落形成测定法测定的细胞杀伤表明，照射组（2）的存活分数几乎与组（1）相同。此外，在组（4）和混合群体组（3）中观察到相似的存活水平。突变诱导，其以HPRT基因座上产生的6-硫代鸟嘌呤抗性克隆的数目来评分，表现出与细胞杀伤效应相同的趋势。因此，有证据表明，在组（2）中未照射的一半细胞中发生了细胞杀伤和突变诱导的旁观者效应。由于林奈明显抑制这些作用，这表明间隙连接介导的细胞间通讯可能在诱导旁观者效应中起重要作用。

We have investigated bystander cellular responses in normal human fibroblasts irradiated with low-fluence ion-beams of low-energy carbon ions. Irradiation dishes were constructed by drilling 6.5mm holes in the center of 35mm-diameter tissue culture dishes and attaching a 2.5μm-thick Mylar film over the bottom of the hole to provide a window for low-energy carbon ion beams. Cells were inoculated onto the Mylar film in the dishes and then irradiated with 6MeV/n carbon ions at a fluence of 2 x 10^5 particles/cm^2. The beam was generated by the Medium Energy Beam Course at the Heavy Ion Medical Accelerator in Chiba (HIMAC). Cells were exposed using 4 different protocols ; (1)Cells were irradiated with all of the cells on the Mylar film ;. (2)Cells were irradiated with half of the cell population on the Mylar film ; (3)Irradiated cells and un-irradiated control cells were pooled in a 1:1 ratio and plated as a single culture ; and (4)Half of the cells were irradiated and treated with a specific inhibitor of gap junction mediated cell-cell communication (40μM lindane). Cell killing, which was assayed with a colony formation assay, showed that the surviving fraction of the irradiated group (2)was almost the same as for group (1).In addition, a similar level of survival was seen in the group (4)and in the mixed population group (3).Mutation induction, which was scored as the number of 6-thioguanine resistant clones arising at the HPRT locus, showed the same trend as the cell killing effect. Thus there is evidence that the bystander effect for both cell killing and mutation induction occurred in the un-irradiated half of the cells in group (2). Since lindane apparently inhibited these effects, this suggests that gap junction mediated cell-cell communication may play an important role in the induction of the bystander effect.