摘要
目的:运用加载miRNA34a的外泌体治疗口腔鳞状细胞癌。方法:采用超速离心的方法从HEK293细胞的培养上清液中提取外泌体,结合透射电子显微镜、纳米粒子追踪分析仪、Western blot实验鉴定外泌体,进而利用共孵育的方法将疏水性修饰的miRNA34a模拟物加载到外泌体中,以构建含miRNA34a的外泌体(exo-miRNA34a),倒置荧光显微镜下观察加载效率。将exo-miRNA34a接种于口腔鳞癌细胞HN6中,利用激光共聚焦显微镜观察HN6细胞对外泌体的摄取,结合CCK-8实验检测HN6细胞增殖的变化。结果:在HEK293细胞培养上清液提取物中观察到40~150 nm大小的小囊泡,囊泡粒径分布峰值为97.9 nm;肿瘤易感基因101蛋白(TSG101)和CD63表达阳性,钙联蛋白(Calnexin)表达阴性。疏水改性的miRNA34a可有效加载到外泌体中,载药率约为47%,载药后外泌体粒径增加,其完整性不受影响。exo-miRNA34a可以进入HN6细胞中,对HN6细胞的增殖具有明显抑制作用(P<0.05)。结论:外泌体介导的miRNA34a可明显抑制口腔鳞癌HN6细胞的增殖。
Objective:To treat oral squamous cell carcinoma through via exosomes loaded with miRNA34a.Methods:First,exosomes ought to be extracted from the culture supernatants of HEK293 cells by means of ultracentrifugation and identified through integrating transmission electron microscopy,nanoparticle tracking analyzer(NTA)with Western blotting experiment,to load miRNA34a mimics modified by hydrophobicity into exosomes by utilizing the method of co-incubation,and to fabricate exo-miRNA34a for loading efficiency observation under an inverted fluorescence microscope.Second,exo-miRNA34a will be inoculated into HN6 cells,so that the up-taking ability of HN6 cells on exosomes will be observed via laser scanning confocal microscope,and the proliferation changes of HN6 cells will be also detected coupled with CCK-8 experiment.Results:Small vesicles of 40-150 nm in size will be observed under a transmission electron microscopy in culture supernatant fluid extract of HEK293 cells,the distribution maxima of particle size of the vesicles is 97.9 nm based on the observation results of NTA,and surface protein,TSG101 and CD63,of exosomes can be detected by the transmission electron microscopy.However,plasmosin-Calnexin turns out negative after detection,which verifies the successful extraction of exosomes.Furthermore,the observation results of fluorescence microscope show that miRNA34a mimics modified by hydrophobicity can be loaded into exosomes effectively during co-incubation,drug loading capacity is 47%approximately.The particle size of exosomes increases after drug loading,and its integrity is not affected.Besides,laser scanning confocal microscope displays that exo-miRNA34a enters into HN6 cells,CCK-8 detection finds that miRNA34a can inhibit the proliferation of HN6 cells obviously(P<0.05).Conclusion:MiRNA34a mediated by exosomes can significantly inhibit the proliferation of HN6 cells in oral squamous cell carcinoma.
作者
邓威
王晨星
金齐尧
毛广艳
孟颖
李怀奇
叶金海
DENG Wei;WANG Chenxing;JIN Qiyao;MAO Guangyan;MENG Ying;LI Huaiqi;YE Jinhai(Jiangsu Key Laboratory of Oral Diseases, Department of Oral and Maxillofacial Surgery, Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing 210029, China)
出处
《口腔生物医学》
2020年第2期92-96,共5页
Oral Biomedicine
基金
国家自然科学基金(81371123)
“科教强卫工程”医学重点人才项目(ZDRCA2016087)
江苏高校优势学科建设工程资助项目(2018-87)。
