支原体污染增加人肿瘤细胞外泌体生成并改变其小RNA表达模式

目的:研究支原体污染对肿瘤细胞外泌体生成的影响,探讨支原体导致的外泌体量和质的变化。方法:应用荧光显微镜及荧光共振能量转移观察支原体污染导致的胞内膜脂质向胞膜再分布及跨细胞传输变化,应用western blotting检测污染细胞上清中外泌体标记物CD81蛋白含量,应用芯片检测污染细胞上清外泌体中小RNA的表达变化模式。结果:支原体污染导致Rab11和胞膜脂质染料信号强度上升2倍以上,提示胞内膜脂质向胞膜再分布增强;支原体污染导致Di O和Di I双染细胞胞内膜泡的FRET指数上升4倍,提示膜脂质由支原体污染细胞向未污染细胞的跨细胞传输增强;污染细胞的上清中外泌体标志物CD81明显升高;污染导致外泌体内小RNA表达模式改变。结论:体外实验显示支原体污染可造成外泌体质和量的改变。

Mycoplasma Contamination Enhances Generation of Exosomes of Human Tumor Cells and Alters the Exosomal miRNA Expression Pattern

ABSTRACT Objective: To investigate the effect of mycoplasma contamination on host cells exosome generation. To study the mycoplasma-induced alterations on exosome in quantity and quality aspects. Methods: Fluorescent microscopy and Fluorescent resonance energy transfer (FRET) were applied for observation of mycoplasma-induced redistribution of intracellular membrane lipid to plasma membrane and intercellular transportation. Western blotting was used to determine the exosomal biomarker CD81 levels in the supernatant of mycoplasma contaminated host cells. MicroRNA chip assay were used to analyze the changes of microRNA expression pattern. Results: Mycoplasma contamination increased fluorescence intensity of Rab 11 and DiI at peripheral cytoplasm region more than 2 folds, indicating enhanced redistribution of intracellular lipid to plasma membrane. Mycoplasma induced 4 folds increase in vesicular FRET index in DiO and DiI double-labeled cells, which indicates intercellular lipid transport is enhanced. And in mycoplasma infected cells supernatant the expression level of exosomal marker CD81 increased remarkably. In addition, exosomal miRNA of mycoplasma-contaminated cells differs from that of mycoplasma free cells. Conclusion: In vitro study demonstrates that mycoplasma contamination may change exosome generation in quantity and quality.