吉林大学学报(医学版) ›› 2018, Vol. 44 ›› Issue (04): 718-723.doi: 10.13481/j.1671-587x.20180405

• 基础研究 • 上一篇    下一篇

线粒体靶向KillerRed诱导的ROS增强辐射对HeLa细胞的增殖抑制作用

李鑫, 马云飞, 唐庚, 韦麒, 纪红池, 田嘉安, 申延男, 王志成   

  1. 吉林大学公共卫生学院 国家卫生健康委员会放射生物学重点实验室, 吉林 长春 130021
  • 收稿日期:2017-12-04 出版日期:2018-07-28 发布日期:2018-07-27
  • 通讯作者: 申延男,副教授,硕士研究生导师(Tel:0431-84995443,E-mail:204197896@qq.com);王志成,副教授,硕士研究生导师(Tel:0431-85619443,E-mail:zhicheng@jlu.edu.cn) E-mail:204197896@qq.com;zhicheng@jlu.edu.cn
  • 作者简介:李鑫(1993-),女,吉林省通化市人,在读医学硕士,主要从事辐射肿瘤学方面的研究。
  • 基金资助:
    吉林省科技厅科技发展计划项目资助课题(20180101305JC);吉林省教育厅科学技术项目资助课题(JJKH20170828KJ,JJKH20180189KJ);吉林省卫计委科研项目资助课题(20165069)

Enhancement of ROS induced by mitochondria-targeted KillerRed in proliferation inhibition of radiation on HeLa cells

LI Xin, MA Yunfei, TANG Geng, WEI Qi, JI Hongchi, TIAN Jiaan, SHEN Yannan, WANG Zhicheng   

  1. NIH Key Laboratory of Radiobiology, School of Public Health, Jilin University, Changchun 130021, China
  • Received:2017-12-04 Online:2018-07-28 Published:2018-07-27

摘要: 目的:构建线粒体靶向KillerRed (KR)重组表达载体,探讨可见光照射诱导活性氧(ROS)生成规律及其增强电离辐射对HeLa细胞的增殖抑制作用。方法:利用基因重组技术构建线粒体靶向重组载体plxsp-flag-Sarm1-KR和plxsp-flag-Sarm1-mCherry。将宫颈癌HeLa细胞分为对照组、plxsp-flag组、plxsp-flag-Sarm1-KR组、4 Gy组、plxsp-flag+4 Gy组和plxsp-flag-Sarm1-KR+4 Gy组。细胞转染24 h后,利用荧光显微镜检测mCherry蛋白和细胞色素C氧化酶Ⅳ(COXⅣ)蛋白表达水平;可见光照射后利用DCFH-DA探针检测平均荧光强度(MFI),表示ROS生成水平;4 Gy X射线照射后,利用CCK-8试剂盒检测细胞增殖活性。结果:构建载体经测序鉴定并与GenBank序列比对,表明线粒体靶向融合表达载体plxsp-flag-Sarm1-KR (mCherry)构建成功。转染HeLa细胞后,荧光显微镜下可见mCherry蛋白与线粒体示踪COXⅣ蛋白具有相同的定位。ROS生成水平,对照组和plxsp-flag组在可见光照射10、30和60 min后掺入探针,掺入探针10、30和60 min后MFI无明显变化(P>0.05); plxsp-flag-Sarm1-KR组MFI呈时间依赖性增加,与对照组比较,plxsp-flag-Sarm1-KR组可见光照射10和30 min后掺入探针,掺入探针60 min后MFI明显增加(P<0.05);可见光照射60 min后掺入探针,掺入探针30和60 min后plxsp-flag-Sarm1-KR组MFI明显降低(P<0.05)。与对照组比较,plxsp-flag组细胞增殖活性无明显变化(P>0.05),10和24 h时plxsp-flag-Sarm1-KR组细胞增殖活性明显降低(P<0.05),10 h时4 Gy组和plxsp-flag+4 Gy组细胞增殖活性明显降低(P<0.01),10、24和48 h时plxsp-flag-Sarm1-KR+4 Gy组细胞增殖活性明显降低(P<0.05或P<0.01);与plxsp-flag-Sarm1-KR组和4 Gy组比较,plxsp-flag-Sarm1-KR+4 Gy组细胞增殖活性明显降低(P<0.05)。结论:成功构建线粒体靶向融合表达载体,所介导的KR蛋白可以诱导ROS产生,并且增强电离辐射对HeLa细胞的增殖抑制作用。

关键词: 细胞增殖, 活性氧, KillerRed, 线粒体, 电离辐射

Abstract: Objective: To construct the recombinant expression vector of mitochondria-targeted KillerRed (KR), and to explore the producing regularity of reactive oxygen species (ROS) induced by visible light exposure, and its enhancement in the proliferation inhibition of radiation on the HeLa cells. Methods: Gene recombination technique was used to build the recombinant vectors plxsp-flag-Sarm1-KR and plxsp-flag-Sarm1-mCherry targeting mitochondria. The cervical cancer HeLa cells were divided into control, plxsp-flag, plxsp-flag-Sarm1-KR, 4 Gy, plxsp-flag + 4 Gy and plxsp-flag-Sarm1-KR + 4 Gy groups. After the vectors were transfected into the cells for 24 h, the expression levels of mCherry and mitochondrial track COXⅣ proteins were determined by fluorescence microscope;DCFH-DA probe was used to detect the mean fluorescence intensity(MFI) to indicate the ROS producing level. After 4 Gy X-ray irradiation,the cell proliferation activity was measured by CCK8 kits. Results: After the vectors were sequenced and identified, the sequence was consistent with that in GenBank, and the results showed the fusion expression vector plxsp-flag-Sarm1-KR(mCherry) was successfully constructed. After the plasmids were transfected into the HeLa cells, under fluorescence microscope, mCherry protein and COXⅣ protein had the same expression location. From ROS producing level, in control and plxsp-flag groups, after the cells were exposed to visble light for 10, 30 and 60 min, the MFI had no obvious changes after the probe was incorporated for 10, 30 and 60 min; but in plxsp-flag-Sarm1-KR group, the MFIs were increased with the time prolongation; compared with control group,the cells were exposed to visble light for 10 and 30 min, and the MFIs were significantly increased after the probe was incorporated for 60 min (P<0.05); when the cells were exposed to visble light for 60 min, the MFIs were significantly decreased after the probe was incorporated for 30 and 60 min (P<0.05). Compared with control group, the A(450) in plxsp-flag group had no obvious change(P>0.05); the proliferation activities were significantly decreased at 10 and 24 h in plxsp-flag-Sarm1-KR group (P<0.05); the proliferation activities were significantly decreased at 10 h in 4 Gy and plxsp-flag + 4 Gy groups (P<0.01); the proliferation activities were significantly decreased at 10, 24 and 48 h in plxsp-flag-Sarm1-KR + 4 Gy group (P<0.05 or P<0.01). The proliferation activity in plxsp-flag-Sarm1-KR + 4 Gy group was lower than those in plxsp-flag-Sarm1-KR and 4 Gy groups (P<0.05). Conclusion:The mitochondria-targeted fusion expression vectors are successfully constructed,and the KR proteins can induce the ROS producing and enhance the inhibitory effect of proliferation of ionizing radiation on the HeLa cells.

Key words: KillerRed, mitochondria, radiation, cell proliferation, reactive oxygen species

中图分类号: 

  • Q754