Bystander cellular effects induced by low-fluence charged particles

我们研究了在低能量碳离子束流照射下正常人成纤维细胞的旁观者细胞反应。在直径为35mm的组织培养皿的中心打6.5mm的孔，在孔的底部贴敷2.5μm厚的Mylar薄膜，为低能碳离子束提供一个窗口，从而构建辐照皿。将细胞接种到培养皿中的Mylar薄膜上，然后用6MeV/n的碳离子以2 × 10^5个粒子/cm^2照射。该束流是由千叶重离子医学加速器（HIMAC）的中能量束流课程产生的。使用4种不同的方法暴露细胞；(1)所有细胞在Mylar薄膜上辐照；(2)用一半的细胞群照射在Mylar薄膜上；(3)照射后的细胞与未照射的对照细胞按1:1的比例混合，作为单个培养物进行电镀；(4)对一半的细胞进行辐射处理，并用间隙连接介导的细胞-细胞通讯特异性抑制剂（40μM林丹）处理。用集落形成试验测定细胞杀伤，结果表明辐照组(2)的存活比例与辐照组(1)几乎相同。此外，在该组(4)和混合种群组(3)中观察到相似的生存水平。突变诱导（以在HPRT位点产生的6-硫鸟嘌呤抗性克隆的数量来衡量）表现出与细胞杀伤效果相同的趋势。因此，有证据表明，在组中未辐照的一半细胞中发生了细胞杀伤和突变诱导的旁观者效应(2)。由于林丹明显抑制了这些效应，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.