Tang Bo, He Da-Wei, Li Dian, Guo Wen-Hao, Zhang Dan, Wei Guang-Hui
Department of Urinary Surgery, Chongqing Key Laboratory of Children Urogenital Development and Tissue Engineering, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation base of Child development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China. E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2018 Mar 20;38(3):334-339. doi: 10.3969/j.issn.1673-4254.2018.03.15.
To investigate the effect of outer membrane vesicles derived from Escherichia coli on proliferation, apoptosis and migration of human neuroblastoma SK-N-SH cells in vitro.
The outer membrane vesicles (OMVs) were obtained from wild-type Escherichia coli with ultracentrifugation method, and the morphology of the OMVs was observed by transmission electron microscopy and the vesicle diameter was determined using MALVERN ZEN3690. Human neuroblastoma SK-N-SH cells were treated with the OMVs at low (100 µg/mL), moderate (500 µg/mL) and high (1000 µg/mL) doses, and 24, 48 and 72 h later, the cell proliferation activity was detected with MTT assay. The expressions of apoptosis-related marker caspase-3 was detected using Western blotting, and TUNEL assay was performed to detect the cell apoptosis. The migration capacity of SK-N-SH cells was evaluated using Transwell migration assay.
The isolated OMVs showed a circular or elliptical hollow structure with double-layer membrane and a diameter range of 30-450 nm. Compared with the control cells, SK-N-SH cells treated with the OMVs showed significantly lowered cell proliferation capacity with enhanced expression of caspase-3. Treatment of the cells with the OMVs resulted in increased cell apoptosis and significantly lowered migration capacity (P<0.05).
The OMVs derived from Escherichia coli can produce cytotoxicity against SK-N-SH cells and might serve as a therapeutic agent for refractory neuroblastoma.
探讨大肠杆菌来源的外膜囊泡对人神经母细胞瘤SK-N-SH细胞体外增殖、凋亡及迁移的影响。
采用超速离心法从野生型大肠杆菌中获取外膜囊泡（OMVs），通过透射电子显微镜观察OMVs的形态，使用马尔文ZEN3690测定囊泡直径。将人神经母细胞瘤SK-N-SH细胞分别用低剂量（100μg/mL）、中剂量（500μg/mL）和高剂量（1000μg/mL）的OMVs处理，24、48和72小时后，采用MTT法检测细胞增殖活性。使用蛋白质免疫印迹法检测凋亡相关标志物caspase-3的表达，并进行TUNEL检测以检测细胞凋亡。采用Transwell迁移试验评估SK-N-SH细胞的迁移能力。
分离得到的OMVs呈现出具有双层膜的圆形或椭圆形中空结构，直径范围为30 - 450nm。与对照细胞相比，用OMVs处理的SK-N-SH细胞增殖能力显著降低，caspase-3表达增强。用OMVs处理细胞导致细胞凋亡增加，迁移能力显著降低（P<0.05）。
大肠杆菌来源的OMVs可对SK-N-SH细胞产生细胞毒性，可能作为难治性神经母细胞瘤的治疗药物。
