肿瘤细胞胞外囊泡DNA特征分析
Analysis of the Characteristics of Extracellular Vesicle DNA of Tumor Cells
陈思宇 1章运生 2裴超柱 2谭拥军2
作者信息
- 1. 中南林业科技大学生命科学与技术学院,长沙 410000
- 2. 湖南大学生物学院,长沙 410000
- 折叠
摘要
从肺腺癌A549细胞培养上清液中分离胞外囊泡(EVs),通过动态光散射(DLS)和透射电镜(TEM)分析得到EVs的大小约为100 nm,并在样品中检测到EVs的特异性标记TSG101.通过琼脂糖凝胶电泳检测发现,胞外囊泡DNA(evDNA)分子片段的大小在12 kb左右.根据evDNA测序数据选择特定evDNA开展PCR试验,实现其有效检测.对EVs进行脱氧核糖核酸酶Ⅰ(DNase I)或ATP依赖的DNA酶(PS酶)处理,证实evDNA以线性方式存在于囊泡外侧.通过末端转移酶(TdT)向evDNA末端添加poly-dA,使用oligo-dT珠对其进行捕获,进一步证明其线性表面分布.此外,建立TetO-DNA/TetR-GFP可视化系统,在荧光显微镜下观察到TetR-GFP蛋白与EVs上TetO-DNA的结合.流式细胞术和PCR试验证实,可用TetR-GFP蛋白实现对携带TetO-DNA EVs的富集.本研究证实了evDNA以线性的形式分布在EVs表面,为进一步研究其生物学功能提供了重要基础.
Abstract
Extracellular vesicles(EVs)were isolated from the culture supernatant of lung adenocarcinoma A549 cells.The size of EVs was characterized as approximately 100 nm via DLS and TEM analysis,with the EV-specific marker TSG101 detected in the samples.The extracted EV DNA(evDNA)showed a predominantly～12 kb size distribution as assessed by agarose gel electro-phoresis.Based on sequencing data,specific evDNA was selected for PCR detection.DNase I or PS nuclease treatment of the EVs confirmed that the evDNA existed on the outside of the vesicle in a linear manner,and poly-dA tails were added to evDNA by TdT enzyme treatment,allowing for their capture by oligo-dT beads and presenting further evidence of their linear surface distribution.Additionally,a TetO-DNA/TetR-GFP visualization system was established to confirm the presence of TetO-DNA in the EVs,which could be visualized by TetR-GFP proteins under fluorescent microscopy.Enrichment of EVs carrying the TetO-DNA was confirmed via flow cytometry and PCR experiments using TetR-GFP protein.These findings collectively suggest that evDNA is distributed linearly on the surface of EVs,providing a basis for further research into their biological functions.
关键词
肺腺癌细胞/胞外囊泡/evDNA/Tet操纵基因-DNA/Tet阻遏蛋白-绿色荧光蛋白Key words
lung adenocarcinoma cells/extracellular vesicles/evDNA/TetO-DNA/TetR-GFP引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据