球形金纳米颗粒对骨髓间充质干细胞自噬流和外泌体分泌的影响

Effect of gold nanoparticle on autophagy flux and exosome secretion of mesenchymal stem cells

目的·探讨球形金纳米颗粒(Au NPs)阻断大鼠骨髓间充质干细胞(MSCs)的自噬流对外泌体分泌的影响。方法·体外培养大鼠MSCs,分别以不同浓度Au NPs(0、15、30、90、150μg/m L)处理细胞,CCK-8法检测细胞活力,以评估Au NPs细胞毒性。细胞分3组:正常对照组、Au NPs组(20μg/m L Au NPs作用72 h)和阳性对照组(5μmol/L氯喹作用4 h)。透射电镜下观察超微结构,超速离心提取细胞上清液外泌体,行纳米颗粒分析仪和电镜检测。共聚焦显微镜观察细胞自噬,Western blotting验证。结果·0~30μg/m L的Au NPs对细胞增殖的影响小。Au NPs组的MSCs外泌体分泌增多,体积减小,LC3和p62表达增高(均P<0.05),自噬小体增多,溶酶体和自噬小体融合受阻。结论·Au NPs阻断MSCs自噬流,调节外泌体分泌。

Objective · To investigate the effect of gold nanoparticles （AuNPs） on exosome secretion of mesenchymal stem cells （MSCs） via blocking autophagy flux. Methods · Rat derived MSCs were treated with AuNPs （0, 15, 30, 90, 150 μg/mL）, and the cell proliferation was detected with CCK-8 assay to evaluate the cytotoxicity of AuNPs. Then, MSCs were divided into normal control group, AuNPs group （20 μg/mL AuNPs for 72 h） and positive control group （5 μmol/L chloroquine for 4 h）. Transmission electron microscopy was used to observe the ultrastructure of MSCs, and exosomes were extracted by ultracentrifugation and tested with nanoparticle analyzer and scan electron microscopy. The autophagy was observed by confocal microscopy and verified by Western blotting. Results · Low concentrations of AuNPs （0 to 30 μg/mL） slightly affected the cell proliferation. MSCs of AuNPs group secreted more abundant and smaller exosomes than those of control group, and expressed significantly more LC3 and p62 （both P〈0.05）. Autophagosomes in AuNPs group were increased and fusion of lysosomes and autophagosomes was blocked. Conclusion · AuNPs block the autophagy flux and regulates the secretion of exosomes.