目的:研究宫颈癌细胞Siha分泌的外泌体(Exosomes)能否介导上皮-间质转化(EMT),从而改变癌前细胞Ect1的体外侵袭能力.方法:差速超速离心法分离Siha-Exo,透射电镜观察形态,Western blot检测标志蛋白表达,共聚焦显微镜观察PKH67染色外泌体的生物学活性.Siha-Exo和PBS分别与Ect1共培养48 h后Western blot检测上皮和间质转化标志蛋白的表达,Transwell侵袭实验检测共培养后Ect1体外侵袭能力.结果:Siha-Exo为直径30~100 nm的盘状囊泡,特异性表达标志性蛋白CD63和CD81,能够被受体细胞Ect1内吞,具备生物学活性.Western blot结果显示,与PBS组相比,Siha-Exo组下调Ect1细胞中上皮标志蛋白E-Cadherin和β-Catenin的表达,上调间质标志蛋白N-Cadherin和Vimentin的表达.Transwell侵袭实验显示,与PBS组相比,Siha-Exo组穿透聚碳酯膜的Ect1细胞数明显增多(P=0.01).结论:Siha-Exo能够介导Ect1发生EMT变化,提高其体外侵袭能力.
Objective:To investigate whether Exosomes secreted by cervical cancer cell lines Siha ( Siha-Exo) have ability to promote Ect1 invasion via epithelial mesenchymal transi-tion ( EMT) in cervical cancer. Methods:Morphology and marker protein of Siha-Exo isolated by different ultracentrifugation were identified by transmission electron microscopy and Western blot. And green fluorescent PKH67-labeled Siha-Exo detected by confocal images for biological activity. Ect1 was co-cultured with Siha-Exo or PBS for 48 hours. The expression of epithelial and mesenchyme markers were examined by Western blot. Transwell chambers were used in vitro invasion assay. Results:Siha-Exo exhibited typical cup-shaped vesicles and a size range of 30-100nm,expressed exosomal marker protein CD63 and CD81,and were biologically uptaked by recipient Ect1. Compared with PBS treated cells,Ect1 treated with Siha-Exo decreased ex-pression of epithelial marker E-Cadherin andβ-Catenin,and increased expression of mesenchy-mal marker N-Cadherin and Vimentin. Meanwhile in Siha-Exo group,numbers of Ect1 penetrat-ing transwell membrane significantly increased in contrast with PBS group ( P=0 . 01 ) . Conclu-sion:Siha-Exo elicited Ect1 to an EMT-like phenotype,thus promoting invasion in vitro.