解聚素-金属蛋白酶17缺失对鼻咽癌细胞活性氧产生及线粒体功能的影响  

Effect of a disintegrin and metalloprotease 17 deletion on production of reactive oxygen species and mitochondrial function in nasopharyngeal carcinoma cells

作　　者：尹永波 李学申 邱金霞 籍玉青 崔静 YIN Yongbo;LI Xueshen;QIU Jinxia;JI Yuqing;CUI Jing(Xingtai People's Hospital,Affiliated Hospital of Hebei Medical University,Xingtai 054000,Hebei Province,China)

机构地区：[1]邢台市人民医院,河北医科大学附属医院,河北邢台054000

出　　处：《中国生物制品学杂志》2023年第5期566-573,共8页Chinese Journal of Biologicals

基　　金：河北省卫生厅科研基金(20150788)。

摘　　要：目的探讨解聚素-金属蛋白酶17(a disintegrin and metalloprotease 17,ADAM17)缺失对鼻咽癌细胞活性氧(reactive oxygen species,ROS)产生及线粒体功能的影响。方法将3组ADAM17基因干扰质粒ADAM17 shRNA及空质粒ADAM17-shRNA-NC转染鼻咽癌细胞株(CNE1),RT-PCR及Western blot法检测干扰效率,选择干扰效果最好的shRNA进行后续试验。CCK-8法检测各组细胞增殖能力;克隆形成试验检测细胞生长情况;流式细胞术检测细胞凋亡情况和线粒体膜电位变化;荧光显微镜检测细胞线粒体氧化损伤产物ROS水平;丙二醛(malondialdehyde,MDA)试剂盒和超氧化物歧化酶(superoxide dismutase,SOD)试剂盒检测细胞氧化应激标志物MDA和SOD含量;Western blot法检测细胞线粒体损伤标记物Bax/Bcl-2、cleaved-caspase 9/caspase 9、cleaved-caspase 3/caspase 3、c-Myc的表达。结果ADAM17-shRNA2组干扰效果最好。与shRNA-NC组相比,ADAM17-shRNA2组细胞的增殖速率明显下降(t=8.964,P=0.036);形成的集落数明显减小(t=10.351,P=0.014);细胞凋亡数量明显增加(t=11.25,P=0.008);细胞内代表ROS水平的荧光强度明显增强;线粒体膜电位明显下降(t=9.233,P=0.013);SOD含量明显降低(t=7.233,P=0.034),MDA含量明显升高(t=7.415,P=0.038);细胞中Bax/Bcl-2、cleaved-caspase 9/caspase 9、cleaved-caspase 3/caspase 3的水平均明显升高(t分别为8.985、9.021和7.789,P分别为0.023、0.011和0.031),c-Myc蛋白表达水平明显减少(t=10.352,P=0.004)。结论干扰ADAM17基因表达通过促氧化诱导SOD水平下降,MDA水平升高,进而缓解细胞膜的氧化损伤;同时促进细胞线粒体ROS表达水平,降低线粒体膜电位,体外抑制细胞增殖,促进细胞凋亡。Objective To investigate the effects of a disintegrin and metalloproteinase 17(ADAM17)deletion on the production of reactive oxygen species(ROS)and mitochondrial function in nasopharyngeal carcinoma(NPC)cells.Methods Three groups of ADAM17 interfering plasmid ADAM17 shRNA and empty plasmid ADAM17-shRNA-NC were transfected into NPC cell line(CNE1)and detected for the interference efficiency by RT-PCR and Western blot to select shRNA with the best interference effect for the follow-up experiments.The cell proliferation was detected by CCK-8 assay,while the cell growth by clone formation test,the apoptosis and changes in mitochondrial membrane potential(MMP)by flow cytometry,the level of mitochondrial oxidative damage product ROS by fluorescence microscope,the contents of oxidative stress markers MDA and SOD by malondialdehyde(MDA)kit and superoxide dismutase(SOD)kit and the expression of mitochondrial damage markers Bax/Bcl-2,cleaved-caspase 9/caspase 9,cleaved-caspase 3/caspase 3 and c-Myc by Western blot.Results ADAM17-shRNA2 group showed the best interference effect.Compared with shRNA-NC group,the proliferation rate of cell in ADAM17-shRNA 2 group decreased significantly(t=8.964,P=0.036);the number of colonies were significantly reduced(t=10.351,P=0.014);the number of apoptosis increased significantly(t=11.25,P=0.008);the fluorescence intensity representing ROS level in cells increased obviously;the mitochondrial membrane potential decreased significantly(t=9.233,P=0.013);the SOD content decreased(t=7.233,P=0.034)and MDA content increased(t=7.415,P=0.038)significantly;the levels of Bax/Bcl-2,cleaved-caspase 9/caspase 9 and cleaved-caspase 3/caspase 3 significantly increased(t=8.985,9.021 and 7.789,P=0.023,0.011 and 0.031,respectively),while the expression of c-Myc proteins significantly decreased(t=10.352,P=0.004).Conclusion Interfering with ADAM17 induced SOD decrease and MDA increase by promoting oxidation,thereby alleviating oxidative damage of cell membrane,which also promoted the expression level of ROS in

关 键 词：解聚素-金属蛋白酶17 鼻咽癌 活性氧 线粒体 

分 类 号：R739.63[医药卫生—肿瘤]