缺氧预处理宫颈癌细胞来源外泌体通过ERK5信号通路影响细胞增殖和凋亡水平的相关机制研究  

Study on the mechanism of the effect of hypoxia preconditioning on the proliferation and apoptosis of cervical cancer cells through ERK5 signaling pathway

作　　者：孙景丽 宋克娟[1] 姚勤[1] 吕腾[1] 刁玉超[1] SUN Jingli;SONG Kejuan;YAO Qin;Lv Teng;DIAO Yuchao(Department of Obstetrics and Gynecology,the Affiliated Hospital of Qingdao University,Qingdao,Shandong 266003,China;Department of Obstetrics and Gynecology,the Central Hospital of Shanxian,Heze,Shandong 274300,China)

机构地区：[1]青岛大学附属医院黄岛分院妇产科,山东青岛266003 [2]单县中心医院妇产科,山东菏泽274300

出　　处：《国际检验医学杂志》2021年第S01期42-46,共5页International Journal of Laboratory Medicine

基　　金：青岛市民生科技计划项目(19-6-46-nsh)。

摘　　要：目的探究缺氧微环境处理宫颈癌细胞后,分泌的外泌体通过ERK5信号通路调节肿瘤细胞的增殖和凋亡的相关研究。方法本研究以宫颈癌细胞系HCC94细胞为研究对象,利用CoCl2试剂构建肿瘤细胞的缺氧微环境。按照处理方法分成2组,即缺氧组和对照组。本研究采用超速离心法进行收集细胞的外泌体,然后将外泌体和HCC94细胞共培养。利用Western blot法检测外泌体标志物CD63分子和Hsp70,Calnexin分子的表达。利用CCK-8试剂盒检测细胞的增殖情况,利用荧光定量聚合酶链反应(RT-PCR)和Western blot法检测ERK5和凋亡相关因子ERK5,Caspase-3,Caspase-9,Bad,Bax和Bcl-2的表达水平。利用AV-PI试剂盒检测细胞的凋亡,利用Transwell试剂盒检测细胞的侵袭能力。结果透射电镜结果显示,外泌体为直径50~150 nm的双层膜囊泡状结构。Western Blot结果显示,外泌体的分子标志物Hsp70,Calnexin和CD63表达阳性。外泌体与宫颈癌HCC94细胞共培养的过程中,可见HCC94细胞可以摄取并内化CY3标记的外泌体。CCK-8检测结果显示,缺氧组外泌体和HCC94细胞共培养体系的增殖能力要明显高于对照组外泌体和HCC94细胞共培养体系(P<0.05)。RT-PCR和Western blot结果显示,缺氧组外泌体和HCC94细胞共培养体系ERK5,Caspase-3,Caspase-9,Bad和Bax的表达水平要明显低于对照组外泌体和HCC94细胞共培养体系,差异有统计学意义(P<0.05),而抗凋亡因子Bcl-2的表达水平要高于对照组外泌体和HCC94细胞共培养体系,差异有统计学意义(P<0.05)。AV-PI试剂盒检测结果显示,缺氧组细胞凋亡率明显低于对照组,两组比较差异有统计学意义(P<0.05)。Transwell试剂盒检测结果显示,缺氧组细胞侵袭能力明显高于对照组,两组比较差异有统计学(P<0.05)。结论缺氧环境下,宫颈癌细胞来源外泌体可以通过ERK5信号通路促进细胞的增殖和侵袭水平,抑制细胞的凋亡。Objective To investigate the effects of hypoxia microenvironment on the proliferation and apoptosis of cervical cancer cells through ERK5 signaling pathway.Methods In this study,cervical cancer cell line hcc94 was used as the research object,and the hypoxia microenvironment of tumor cells was constructed by CoCl2 reagent.According to the treatment methods,they were divided into two groups:hypoxia group and control group.In this study,the exosomes were collected by ultracentrifugation and co cultured with hcc94 cells.Western blot was used to detect the expression of CD63,HSP70 and calnexin.CCK-8 kit was used to detect cell proliferation.RT-PCR and Western blot were used to detect the expression of ERK5 and apoptosis related factors Caspase-3,Caspase-9,BAD,Bax and bcl-2.AV-PI kit was used to detect cell apoptosis and Transwell kit was used to detect cell invasion.Results The results of transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50-150 nm.The result of the Western blot showed that Hsp70,calnexin and CD63 were positive in exosomes.During the co-culture of exosomes with HCC94 cells,hcc94 cells could absorb and internalize Cy3 labeled exosomes.The results of the CCK-8 assay showed that the proliferation ability of HCC94 cells co-cultured with exosomes in hypoxia group was significantly higher than that in control group,and the difference was statistically significant(P<0.05).The results of the RT-PCR and Western blot showed that the expression levels of ERK5,Caspase-3,caspase-9,BAD and Bax in the co-culture system of exosomes and HCC94 cells in hypoxia group were significantly lower than those in control group,and the difference was statistically significant(P<0.05),while the expression level of anti apoptotic factor Bcl-2 was higher than that of the control group,and the difference was statistically significant(P<0.05).The results of AV-PI kit showed that the apoptosis rate of hypoxia group was significantly lower than that of control group,and the difference was

关 键 词：缺氧 宫颈癌 外泌体 增殖 凋亡 

分 类 号：R73[医药卫生—肿瘤]