大肠杆菌胞外囊泡对人神经母细胞瘤SK-N-SH细胞增殖、凋亡和迁移的影响

目的研究大肠杆菌胞外囊泡（E.coli-OMVs）对人神经母细胞瘤SK-N-SH细胞增殖、凋亡和迁移的影响。方法采用差速离心法提取大肠杆菌胞外囊泡，观察囊泡形态并测定囊泡粒径范围。随机分为4组：正常对照组、胞外囊泡低剂量组（100 μg/mLE.coli-OMVs）、胞外囊泡中剂量组（500 μg/mL E.coli-OMVs）和胞外囊泡高剂量组（1000 μg/mL E.coli-OMVs）。MTT检测各组细胞增殖能力；TUNEL和Western blot分别检测各组细胞凋亡情况及凋亡相关蛋白caspase3的表达；Transwell检测各组细胞迁移能力。结果透射电镜下E.coli-OMVs呈双层膜、中空、圆形或椭圆形，其内含低密度或高密度物质，具有明显的异质性，粒径分析结果表明其直径分布在30~450 nm。与对照组相比，经E.coli-OMVs干预后，SK-N-SH细胞增殖能力显著降低；细胞凋亡增加，caspase3蛋白表达水平明显上调；而迁移能力显著降低（P<0.05）。结论E.coli-OMVs对SK-N-SH细胞具有直接杀伤作用，有望成为难治性神经母细胞瘤的治疗药物。

Effect of outer membrane vesicles derived from Escherichia coli on proliferation,apoptosis and migration of human neuroblastoma SK-N-SH cells in vitro

Objective 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. Methods The outer membrane vesicles (OMVs) wereobtained from wild-type Escherichia coli with ultracentrifugation method, and the morphology of the OMVs was observed bytransmission electron microscopy and the vesicle diameter was determined using MALVERN ZEN3690. Humanneuroblastoma 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 apoptosisrelatedmarker 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. Results The isolated OMVs showed acircular or elliptical hollow structure with double-layer membrane and a diameter range of 30-450 nm. Compared with thecontrol cells, SK-N-SH cells treated with the OMVs showed significantly lowered cell proliferation capacity with enhancedexpression of caspase-3. Treatment of the cells with the OMVs resulted in increased cell apoptosis and significantly loweredmigration capacity (P<0.05). Conclusion The OMVs derived from Escherichia coli can produce cytotoxicity against SK-N-SHcells and might serve as a therapeutic agent for refractory neuroblastoma.