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plxspflag-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、4Gy组、plxsp-flag+4Gy组和plxsp-flag-Sarm1-KR+4Gy组。细胞转染24h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;4Gy 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60min后掺入探针,掺入探针10、30和60min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30min后掺入探针,掺入探针60min后MFI明显增加(P<0.05);可见光照射60min后掺入探针,掺入探针30和60min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24h时plxsp-flag-Sarm1-KR组细胞增殖活性明显降低(P<0.05),10h时4Gy组和plxsp-flag+4Gy组细胞增殖活性明显降低(P<0.01),10、24和48h时plxsp-flagSarm1-KR+4Gy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4Gy组比较,plxsp-flag-Sarm1-KR+4Gy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。
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 for24 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 COXⅣprotein 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
作者
李鑫
马云飞
唐庚
韦麒
纪红池
田嘉安
申延男
王志成
LI Xin;MA Yuntei;TANG Geng;WEI Qi;JI Hongchi;TIAN Jiaan;SHEN Yannan;WANG Zhicheng(NIH Key Laboratory of Radiobiology,School of Public Health,Jilin University,Changchun 130021,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2018年第4期718-723,891,共7页
Journal of Jilin University：Medicine Edition
基金
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)
