【摘要】 目的研究100 MeV质子照射对人宫颈癌HeLa细胞线粒体氧化损伤及其作用机制。方法采用中国原子能科学研究院100 MeV强流质子回旋加速器照射人宫颈癌HeLa细胞,剂量率为0.8 Gy/min。按照射剂量分为未照射组(0 Gy)、低剂量照射组(0.5 Gy)和高剂量照射组(8 Gy),分别于照射后24、48和72 h采用CCK-8法检测细胞增殖情况,流式细胞术检测细胞凋亡变化;DCFH-DA标记法检测照射后12～72 h活性氧(ROS)含量动态变化;线粒体呼吸链复合体Ⅰ检测试剂盒检测照射后24和48 h线粒体呼吸链复合体Ⅰ活性;mRNA芯片杂交检测分析8 Gy质子照射12和24 h后HeLa细胞的差异表达基因。结果与未照射组相比,低剂量照射组细胞增殖、细胞凋亡、ROS生成以及线粒体呼吸链复合体Ⅰ活性均无明显改变。高剂量照射组在照射后72 h细胞增殖显著降低;24、48和72 h细胞凋亡呈时间依赖性增加;细胞ROS含量在照射后16～24 h明显增加,36 h后逐渐下降;照射后24和48 h,细胞线粒体呼吸链复合体Ⅰ活性降低。KEGG分析提示,8 Gy质子照射24 h后诱导表达差异变化明显的基因主要集中在丝裂原活化蛋白激酶(MAPK)信号通路、PI3K/Akt信号通路和过氧化物酶体增殖物(PPAR)激活受体信号通路等。结论 100 MeV质子高剂量照射可抑制人宫颈癌HeLa细胞增殖,促进凋亡,且此改变可能与细胞氧化应激和线粒体呼吸链复合体Ⅰ活性受到抑制有关。更多还原

【Abstract】 Objective To study the mechanism of oxidative damage induced by 100 MeV proton irradiation in human cervical cancer HeLa cells. Methods A 100 MeV high-current proton cyclotron was used at the dose rate of 0.8 Gy/min in China Institute of Atomic Energy. Cells were divided into three groups:the non-irradiated group(0 Gy),low-dose proton irradiation group(0.5 Gy)and high-dose proton irradiation group(8 Gy). Cell proliferation activity was evaluated by CCK-8 assay and apoptosis rate was analyzed by flow cytometry in 24,48 and 72 h after irradiation. Reactive oxygen species(ROS)contents were detected by DCFH-DA labeling within 12-72 h after irradiation and mitochondrial respiratory chain complex Ⅰ activity was detected using complex Ⅰ enzyme activity assay kit within 24-48 h after irradiation. Gene expres-sion was analyzed at 12 and 24 h after 8 Gy proton irradiation by mRNA chip hybridization. Results Compared with thenon-irradiated group,changes in cell proliferation,apoptosis,reactive oxygen species(ROS)content,and mitochondrialrespiratory chain complex Ⅰ activity in the low-dose 0.5 Gy proton-irradiated group were not significant. In the high-dose 8 Gy proton irradiation group,cell proliferation was inhibited at 72 h after irradiation. Apoptosis in the high dose 8 Gy protonirradiation group increased at 24,48 and 72 h after irradiation in a time-dependent manner. It was found that cellular ROSincreased at 16 h after 8 Gy proton irradiation,increased significantly at 24 h,and gradually decreased after 36 h. Mito-chondrial respiratory chain complex Ⅰ activity was significantly decreased at 24 and 48 h after 8 Gy proton irradiation.KEGG analysis showed that differentially expressed genes were mainly involved in the mitogen-activated protein kinase(MAPK)signal pathway,PI3 K/Akt signal pathway,and peroxisome proliferator activated receptor(PPAR)signaling path-way after high dose 8 Gy proton irradiation. Conclusion High dose proton irradiation can inhibit HeLa cell proliferationand promote apoptosis,which may be due to the increase in oxidative stress and the inhibition of mitochondrial respiratorychain complex Ⅰ activity.更多还原