Laser controlled singlet oxygen generation in mitochondria to promote mitochondrial DNA replication in vitro.
Laser controlled singlet oxygen generation in mitochondria to promote mitochondrial DNA replication in vitro.
复制标题
激光控制线粒体中单线态氧的产生促进线粒体 DNA 体外复制
DOI：
10.1038/srep16925
复制
发表时间：
2015-11-18
影响因子：
4.6
通讯作者：
Zhang H
中科院分区：
文献类型：
--
作者：
Zhou X;Wang Y;Si J;Zhou R;Gan L;Di C;Xie Y;Zhang H
研究表明，一定水平的活性氧（ROS）可以促进线粒体DNA（mtDNA）的复制。然而，目前还不清楚是否是线粒体ROS刺激mtDNA复制，这需要进一步研究。在这里，我们采用了光动力系统，以实现控制线粒体单线态氧（1 O2）的产生。用5-氨基乙酰丙酸（ALA）孵育的HeLa细胞暴露于激光照射以诱导线粒体内1 O2的产生。ALA处理的细胞在低剂量的630 nm激光照射后检测到mtDNA拷贝数增加。刺激的mtDNA复制与线粒体1 O2的产生直接相关，使用特定的ROS清除剂进行验证。线粒体DNA复制受线粒体转录因子A（TFAM）和线粒体DNA聚合酶γ的调控。线粒体DNA控制区的修改诱导的1 O2产生的线粒体。照射后ALA处理的细胞中检测到8-氧代鸟嘌呤（8-oxoG）水平显著增加。ALA处理后的HeLa细胞生长刺激和G1-S细胞周期转换也被观察到。这些细胞反应可能是由于在线粒体中检测到的ROS产生的第二波。总之，我们描述了一种可控的体外诱导mtDNA复制的方法。
Reports have shown that a certain level of reactive oxygen species (ROS) can promote mitochondrial DNA (mtDNA) replication. However, it is unclear whether it is the mitochondrial ROS that stimulate mtDNA replication and this requires further investigation. Here we employed a photodynamic system to achieve controlled mitochondrial singlet oxygen (1O2) generation. HeLa cells incubated with 5-aminolevulinic acid (ALA) were exposed to laser irradiation to induce 1O2 generation within mitochondria. Increased mtDNA copy number was detected after low doses of 630 nm laser light in ALA-treated cells. The stimulated mtDNA replication was directly linked to mitochondrial 1O2 generation, as verified using specific ROS scavengers. The stimulated mtDNA replication was regulated by mitochondrial transcription factor A (TFAM) and mtDNA polymerase γ. MtDNA control region modifications were induced by 1O2 generation in mitochondria. A marked increase in 8-Oxoguanine (8-oxoG) level was detected in ALA-treated cells after irradiation. HeLa cell growth stimulation and G1-S cell cycle transition were also observed after laser irradiation in ALA-treated cells. These cellular responses could be due to a second wave of ROS generation detected in mitochondria. In summary, we describe a controllable method of inducing mtDNA replication in vitro.