bEnd.3细胞来源外泌体促成骨效应的体外研究

Osteogenic effect of exosomes derived by bEnd.3 cells:an in vitro study

目的观察小鼠脑微血管内皮细胞bEnd.3来源的外泌体对骨髓间充质干细胞(BMSCs)成骨效应的影响。方法通过超高速离心法提取bEnd.3细胞来源的外泌体(b-Exo),利用透射电子显微镜(TEM)、纳米粒径追踪分析(NTA)和外泌体蛋白标志物蛋白质印迹法(Western blot)实验鉴定所提取的外泌体。与磷酸盐缓冲液(PBS)对照,使用茜素红染色检测其对BMSCs基质矿化,反转录-聚合酶链反应(RT-PCR)检测成骨分化相关基因Runx2、Osteopontin、Osteocalcin表达的影响。组间比较采用t检验。结果TEM下观察到b-Exo具有典型囊泡样结构,NTA显示b-Exo浓度为7.5×10^(10)颗粒/ml,直径为(100.0±7.3)nm。b-Exo可显著增强BMSCs基质矿化,b-Exo组成骨分化相关基因核心结合蛋白因子2(Runx2)、骨桥蛋白(Osteopontin)和骨钙蛋白(Osteocalcin)的表达显著高于对照组(8.28±0.34比3.71±0.40,t=4.974,P<0.01;9.19±1.91比4.09±0.88,t=5.562,P<0.01;10.31±1.67比5.32±0.31,t=5.432,P<0.01),差异均有统计学意义。结论bEnd.3细胞来源的外泌体能够促进BMSCs的成骨分化,有望应用于骨再生和骨质疏松症的无细胞治疗。

Objective To investigate the osteogenic effect of exosomes derived by mouse brain microvascular endothelial cell line bEnd.3 on bone marrow mesenchymal stem cells(BMSCs).Methods Ultracentrifuge was used to extract the exosomes derived by brain-derived endothelial cells.3(bEnd.3)cells bEnd.3 cells derived exosomes(b-Exo).Transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blotting were use to characterize the exosomes.Alizarin red staining was used to detect the osteogenic effects of the exosomes.The relative messenger ribonucleic acid(mRNA)expression of osteogenic-related genes runt-related transcription factor 2(Runx2),osteopontin and osteocalcin was detected by reverse transcriptase-polymerase chain reaction(RT-PCR).Results TEM showed that the particles presented a typical vesicle-like shape.NTA manifested the concentration of Exo was 7.5×10^(10)particles/ml and the diameter was(100.0±7.3)nm.The b-Exo could enhance the BMSCs matrix mineralization and increase the mRNA expression of osteogenic-related genes Runx2,osteopontin and osteocalcin compared to the controls(8.28±0.34 vs.3.71±0.40,t=4.974,P<0.01;9.19±1.91 vs.4.09±0.88,t=5.562,P<0.01;10.31±1.67 vs.5.32±0.31,t=5.432,P<0.01).Conclusion The b-Exo could promote the osteogenic performance of BMSCs and may be applied to the bone regeneration and the cell-free treatment of osteoporosis in the near future.