摘要
目的外泌体与肿瘤细胞的发生发展密切相关,但具体机制不详。文中旨在探讨非小细胞肺癌A549细胞来源外泌体对正常肺上皮BEAS-2B细胞紧密连接蛋白及骨架重塑的影响。方法超速离心法提取A549细胞exosome,透射电镜进行形态学观察,Western blot鉴定表面标志物CD9、CD63。实验分实验组和对照组,实验组为BEAS-2B细胞中加入终浓度为50μg/m L的exosome,对照组为BEAS-2B细胞中加入等量的PBS液,通过免疫荧光实验、Western blot实验、q PCR实验检测exosome对BEAS-2B细胞紧密连接蛋白ZO-1和Occludin蛋白及mRNA表达的影响,phalloidin-FITC荧光染色实验检测exosome对BEAS-2B细胞骨架重塑的影响,Transwell侵袭实验检测exosome处理BEAS-2B后对A549细胞侵袭BEAS-2B细胞能力的影响。结果免疫荧光实验表明,与对照组相比,实验组的ZO-1及Occuldin的表达明显减少;Western blot实验也表明exosome处理BEAS-2B后可显著减少ZO-1和Occuldin蛋白的表达(P<0.05)。q PCR实验表明,与对照组相比,外泌体处理BEAS-2B后可明显下调紧密连接蛋白ZO-1(1.00±0.00 vs 0.45±0.04)和Occuldin mRNA(1.00±0.00 vs 0.52±0.08)水平,差异有统计学意义(P<0.01)。phalloidin-FITC检测结果表明,与对照组相比exosome直接处理后BEAS-2B细胞后F-actin重聚明显增多。Exosome处理BEAS-2B后A549穿过微孔膜的细胞数量[(22.0±4.0)个]明显高于对照组[(10.6±4.5)个],差异有统计学意义(P<0.05)。结论 A549细胞来源exosome可通过下调BEAS-2B细胞紧密连接蛋白ZO-1、Occludin的表达,促进BEAS-2B细胞骨架重塑,最终破坏BEAS-2B细胞对肿瘤细胞A549的屏障作用。
Objective A large number of studies have shown that the exosome is closely related to the occurrence and devel -opment of tumor cells , but the specific mechanism is still unknown .The study was to investigate the effects of A 549-derived exosome on tight junction protein and cytoskeleton remodeling in normal lung epithelial BEAS-2B cells. Methods A549-derived exosome were isolated by ultracentrifugation , the morphology of which was observed by transmission electron microscope .Western blot analysis was used to examine the surface markers of CD 9 and CD63.Immunofluorescence assay, western blot assay and qPCR assay were applied to detect the effects of exosome on tight junction protein ZO-1 and occludin mRNA expression in BEAS-2B cell.Phalloidin-FITC staining experiment was used to detect the effects of exosome on the cytoskeleton remodeling of BEAS-2B cells.The invasiveness of A549 to BEAS-2B was detected by Transwell invasion test . Results Typical vesicles were observed under electron microscope and exosome markers CD 9 and CD63 expression were detected.The expression levels of ZO-1, occludin pro-tain （P〈0.05） and mRNA （P〈0.01） were decreased in exosome-treated BEAS-2B cells, and the cytoskeleton remodelled .Transwell invasion test showed that the number of A 549 cells passing through the microporous membrane （22±4） after exosome treatment BEAS-2B was significantly higher than that of the control group （10.6±4.5） （P〈0.05）. Conclusion A549-derived exosome can promote the cytoskeleton remodeling of BEAS-2B cells by down-regulating the expression of ZO-1 and occludin in BEAS-2B cells, aiming to de-stroy the barrier of BEAS-2B cell to tumor cell A549.
出处
《医学研究生学报》
CAS
北大核心
2017年第10期1035-1040,共6页
Journal of Medical Postgraduates
基金
四川省教育厅科研创新项目(17TD0012)
四川省卫生和计划生育委员会科研课题(16PJ112
16ZD040)
四川省医学会科研项目(Q16015)
四川省医学会科研课题(S15019)
成都医学院科研创新团队(CYTD15-03)
关键词
A549
紧密连接蛋白
骨架重塑
侵袭
Exosome
BEAS-2B
Exosome
BEAS-2B
Tight junction protein
Cytoskeleton remodeling
Invasion
