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目的观察K562细胞分泌的外泌体exosomes。构建CD63与绿色荧光蛋白融合蛋白载体,并在K562细胞中表达。方法采用梯度离心的方法分离K562细胞培养上清中的exosomes,并用扫描电镜观察。构建pEGFP-N1-CD63重组质粒,以不同方式转染至K562细胞,观察表达效率的差异。结果质粒pEGFP-N1-CD63经测序鉴定,通过激光共聚焦显微镜观察,能够在K562细胞中表达。Amaxa核转染的效率高于脂质体转染的方式。结论成功构建pEGFP-N1-CD63质粒并在K562细胞中表达,可对exosomes进行示踪,为后续实验奠定基础。
Objective To observe exosomes secreted by K562 cells. CD63 and green fluorescent protein fusion protein vector were constructed and expressed in K562 cells. Methods The exosomes in K562 cell culture supernatant were separated by gradient centrifugation and observed by scanning electron microscopy. The recombinant plasmid pEGFP-N1-CD63 was constructed and transfected into K562 cells in different ways to observe the difference in expression efficiency. Results The plasmid pEGFP-N1-CD63 was identified by sequencing and was expressed in K562 cells by laser confocal microscopy. Amaxa nuclear transfection is more efficient than liposome transfection. Conclusion The plasmid pEGFP-N1-CD63 was successfully constructed and expressed in K562 cells. The exosomes could be traced and laid the foundation for further experiments.