线粒体靶向KillerRed增强辐射诱导HeLa细胞自噬作用及其机制

目的：探讨线粒体靶向KillerRed（mtKR）增强辐射诱导HeLa细胞线粒体失能及细胞自噬作用，并阐明其相关分子机制。方法：线粒体靶向质粒PTEN诱导激酶1（Pink1）-mtKR转染HeLa细胞后，进行可见光和4 Gy X射线照射，实验分为对照组、空载体组、Pink1-mtKR组、对照+4 Gy X射线照射组、空载体+4 GyX射线照射组和Pink1-mtKR+4 GyX射线照射组。X线照射后24 h，采用流式细胞术检测线粒体膜电位（MMP）和细胞自噬率，采用生化试剂盒检测线粒体呼吸链复合物Ⅰ和Ⅲ水平，采用Western blotting法检测自噬分子P62、Pink1、帕金蛋白（parkin）和线粒体外膜转换酶20（Tom20）的表达量。结果：Pink1-mtKR瞬时转染HeLa细胞后，给予可见光联合4 Gy X射线照射，与对照组比较，Pink1-mtKR组细胞MMP、线粒体呼吸链复合物Ⅰ和Ⅲ水平均明显降低（P<0.05），细胞自噬率明显升高（P<0.05），Pink1-mtKR+4 GyX射线照射组细胞MMP、线粒体呼吸链复合物Ⅰ和Ⅲ水平进一步降低（P<0.05），自噬率进一步升高（P<0.05）。与对照组比较，Pink1-mtKR组和Pink1-mtKR+4 Gy X射线照射组细胞总蛋白中Pink1和parkin蛋白表达量无明显变化，而P62蛋白表达量增加，Pink1-mtKR组和Pink1-mtKR+4 Gy X射线照射组细胞线粒体蛋白中Pink1、parkin和Tom 20蛋白表达量均明显增加。结论：mtKR可增强辐射诱导的线粒体失能和自噬，其机制可能涉及到Pink1/parkin自噬通路的调控。

Enhancement of mitochondria-targeted KillerRed in autophagy caused by radiation in HeLa cells and its mechanism

Objective: To explore the enhancement of mitochondira-targeted KillerRed(mtKR) on the mitochondrial dysfunciton and autophagy caused by radiation in the HeLa cells, and to clarify the relative molecular mechanisms. Methods: After mitochondria-targeted expression vectors Pink-mtKR were transfected into the HeLa cells, the cells were irradiated by visible light and 4 Gy X-ray. The cells were divided into control, empty vector, Pink1-mtKR, control + 4 Gy X-ray irradiation, empty vector + 4 Gy X-ray irradiation and Pink1-mtKR+4 Gy X-ray irradiation groups. After the cells were irradiated with X-ray for 24 h, the mitochondrial membrane potentials (MMP) and the autophgic rates were detected by flow cytometry, the levels of mitochondrial respiratory chain complex Ⅰ and Ⅲ were measured by biochemical assay,and the expression amounts of P62, Pink1, parkin and Tom20 proteins were measured by Western blotting method. Results: After Pink1-mtKR plasmids were transfected into the HeLa cells, the cells were irradiated by visible light and 4 Gy X-ray; compared with control group, the MMP and the levels of mitochondrial respiratory chain complex Ⅰand Ⅲ in Pink1-mtKR group were significantly decreased (P<0.05), and the autohpagic rate was significantly increased (P<0.05); the MMP and the levels of mitochondrial respiratory chain complex Ⅰ and Ⅲ in Pink1-mtKR + 4 Gy X-ray irradiation group were decreased obviously (P<0.05), and the autohpagic rate was significantly increased(P<0.05). Compared with control group, the expression amounts of Pink1 and parkin proteins in total protein of the cells in Pink1-mtKR group and Pink1-mtKR+4 Gy X-ray irradiation group had no obvious changes, but the expression amounts of the P62 protein was increased; the expression amounts of Tom20, Pink1 and parkin in mitochondrial protein in Pink1-mtKR group and Pink1-mtKR+4 Gy X-ray irradiation group were increased obviously. Conclusion: MtKR may enhance the mitochondrial dysfunction and autohpagy caused by radiation, and its mechamism may be related to the regulation of Pink1/parkin autophgy pathway.