乳腺癌细胞外泌体的分离与鉴定

Isolation and identi cation of exosomes from breast cancer cells

目的:从乳腺癌MDA-MB-231细胞中分离外泌体并分析其来源及分子生物学特征。方法:采用多步差速离心的方法从乳腺癌MDA-MB-231细胞中分离外泌体,并在原子力显微镜下观察其形态;采用RT-PCR法检测其特异性的微小RNA(micro RNA,miR)-21的表达;蛋白质印迹法检测其标记物Flotillin-1蛋白的表达。结果:从乳腺癌MDA-MB-231细胞中分离获得外泌体,形态为圆形或椭圆形,大小不一,直径约在30～100nm;RT-PCR检测结果显示,外泌体中有miR-21的表达;蛋白质印迹法检测结果显示,外泌体中有Flotillin-1蛋白的表达。结论:多步差速离心是一种较为简单有效的外泌体提取方法。从形态及分子生物学特征方面证实,本研究中所获得的提取物正是乳腺癌MDA-MB-231细胞释放的外泌体。

Objective： To isolate exosomes from human breast cancer cell line MDA-MB-231 and to analyze their origin and molecular biological characteristics. Methods： Exosomes were isolated from MDA-MB-231 cells by multi-step differential centrifugation and further examined with atomic force microscope. The expression of marker miR （microRNA）-21 was measured by RT-PCR （reverse transcription-PCR）, and the expression of marker protein Flotillin-1 protein was validated by Western blotting. Results： The exosomes were spherical or oval-shaped, with a diameter between 30 to 100 nm, and they were positive in the expressions of miR-21 and Flotillin-1 proteins. Conclusion： The multi-step differential centrifugation is a simple method which can be used to isolate exosomes efficiently. Based on the morphological and molecular features, the extracts obtained from MDA-MB-231 cells are exosomes exactly.