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贾美娥; 李志勇; 徐凯; 王怡衡; 于菲; 何祥一
华西口腔医学杂志, 06/2021, Volume: 39, Issue: 3Journal Article
R739.8; 目的 通过将Cal27外泌体(Cal27-exo)与正常人牙龈成纤维细胞(NHGFs)共培养,测定共培养后NHGFs+Cal27-exo的增殖迁移能力及相关蛋白表达情况,探讨Cal27-exo对NHGFs的活化及生物学行为的影响.方法 采用超速离心法提取Cal27-exo,并用蛋白免疫印迹(Western blot)、透射电子显微镜以及粒径分析对外泌体进行鉴定.将Cal27-exo与NHGFs共培养,检测NHGFs对Cal27-exo的摄取情况,用划痕实验和CCK8检测NHGFs+Cal27-exo的迁移和增殖能力,用实时荧光定量聚合酶链反应检测NHGFs活化相关蛋白金属基质蛋白酶9(MMP-9)、成纤维细胞活化蛋白(FAP)、α平滑肌肌动蛋白(αSMA)和转化生长因子β(TGF-β)的mRNA表达.结果 超速离心法提取的Cal27-exo中Alix和CD63表达阳性;透射电子显微镜示Cal27-exo呈类圆形双层膜性囊泡;粒径检测示Cal27-exo直径为30~150 nm.共培养后Cal27-exo进入NHGFs内,划痕实验结果示NH-GFs+Cal27-exo的迁移能力强于NHGFs,CCK8结果示NHGFs+Cal27-exo的增殖活力强于NHGFs.实时荧光定量聚合酶链反应结果显示,与NHGFs相比,NHGFs+Cal27-exo的MMP-9表达上调,TGF-β、αSMAmRNA表达下调(P<0.05).结论 NHGFs+Cal27-exo的增殖活性和迁移能力明显增强,相关蛋白的mRNA水平改变.Cal27-exo能活化NHGFs,对肿瘤的侵袭转移有潜在意义.
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