肝癌细胞来源的exosomes提取方法的改进及电镜观察

ISOLATION METHOD IMPROVEMENT OF EXOSOMES DERIVED FROM HEPATOCARCINOMA CELL AND OBSERVATION WITH ELECTRON MICROSCOPE

目的：分离提纯两种肝癌细胞HepG2细胞和Hep3B细胞来源的exosomes，并在透射电镜下观察exosomes的形态特征，为肿瘤免疫治疗寻找肿瘤新抗原提供实验材料。方法：采用离心超滤联合蔗糖密度梯度离心等方法分离出HepG2和Hep3B细胞释放exosomes，经透射电镜观察其超微结构。结果：HepG2和Hep3B细胞分泌释放的exosomes为直径50-100nm的膜性微囊结构，圆形或椭圆形，腔内为低电子密度成分。结论：离心超滤和蔗糖密度梯度离心法分离提纯exosomes方法是较为简便高效，对肿瘤细胞培养上清提纯exosomes而言具有可行性。

Objective：Isolating and purifing the exosomes which were derived from HepG2 cell and Hep3 B ceU to investigate the possibility of using these exosomes as a kind of tumor vaccine. Methods： Exosomes were purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation, and then observed their ultrastructures by electron microscopy. Results：Transmission electron microscopy revealed that the exosomes from HepG2 cell and Hep3B cell culture supematants were 50 to 100nm membrane vesicles. They were round or elliptic in shape. Cavum had low electrondensity components. Conclusion：Uhrafiltration centrifugation technique is a convenient and useful method for isolating exosomes, and it can be used to purify tumor cell-secreted exosomes.