外泌体—电离辐射诱导旁效应的另一种机制

采用条件培养液转移的方法,以微核形成和克隆形成为终点,检测X-射线在H460非小细胞肺癌细胞中诱导经培养液介导的旁效应;采用差速离心法从未受照射和受照H460细胞培养液中提取、纯化外泌体,用透射电镜观察其形态,激光粒度仪分析其粒径分布,并用Western blot检测其标志蛋白hsp90β的表达。通过荧光探针共定位实验观察外泌体进入接收细胞的情况,采用结晶紫实验检测外泌体对接收细胞增殖的影响。结果表明:X-射线可在H460细胞中诱导经培养液介导的旁效应,表现为旁效应细胞的微核增加和克隆存活率下降;未受照射和受照细胞均分泌外泌体,但不同条件下细胞分泌外泌体的尺寸分布不同;当将外泌体加入到接收细胞时,外泌体可能通过膜融合的方式很快进入接收细胞,进而促进接收细胞的增殖;受照射H460细胞分泌的外泌体不影响接收细胞的克隆存活率,但增加接收细胞的微核形成率,并且这种现象在用RNA酶处理外泌体后消失。以上结果表明,受照射H460细胞分泌的外泌体可能是介导电离辐射旁效应的机制之一,并且其中RNA可能是介导电离辐射诱导旁效应的一种重要信号分子。

Exosomes--another underlying mechanism of radiation-induced bystander effects

Using micronucleus formation and clonogenic cell survival as endpoints, medium-mediated bystander effects in H460 non-small cell lung cancer cells induced by X-ray irradiation was studied adopting medium transfer method. Differential centrifugation method was used to isolate and purify exosomes from the media of unirradiated and irradiated H460 cells. The morphology of exosomes was observed using transmission electron microscope, the size distribution of exosomes was measured using Zetasizer Nano ZS 90 size detector, and the expression of a marker of exosomes, hsp90β, was detected by Western blot. Fluorescence probes were applied to investigate the internalization of exosomes into recipient cells. The effects of exosomes on the proliferation of recipient cells were assessed using crystal violet assay. The data showed that X-rays could induce medium-mediated bystander effects in H460 cells manifesting as an increase in micronucleus formation and a decrease in clonogenic cell survival of bystander ceils. Both unirradiated and irradiated H460 cells secreted exosomes, but with different size distribution. When incubated with recipient cells, exosomes could be internalized quickly into recipient cells probably through membrane fusion, promoting proliferation in recipient cells. The exosomes secreted by irradiated cells had no effects on the clonogenic cell survival rate of recipient cells, while they could induce an increase in the frequency of micronucleus formation in recipient cells, which could be abolished by the pretreatment of exosomes with RNase. These results suggest that exosome secreted by irradiated cells might be one of the mechanisms underlying RIBEs, and RNAs in exosomes might play an important role.