肿瘤细胞胞外囊泡DNA特征分析  

Analysis of the Characteristics of Extracellular Vesicle DNA of Tumor Cells

作　　者：陈思宇 章运生[2] 裴超柱 谭拥军[2] CHEN Siyu;ZHANG Yunsheng;PEI Chaozhu;TAN Yongjun(College of Life Science and Technology,Central South University of Forestry and Technology,Changsha 410000,China;College of Biology,Hunan University,Changsha 410000,China)

机构地区：[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

出　　处：《激光生物学报》2024年第2期151-159,共9页Acta Laser Biology Sinica

基　　金：国家自然科学基金项目(81773169)。

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。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.

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 

分 类 号：Q71[生物学—分子生物学]