（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）

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。

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

中图分类号：Q71                                  文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.02.007

(1. College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410000, China; 

2. College of Biology, Hunan University, Changsha 410000, China)

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 electrophoresis. 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.

Key words: lung adenocarcinoma cells; extracellular vesicles; evDNA; TetO-DNA; TetR-GFP

（Acta Laser Biology Sinica, 2024, 33(2): 151-159）