Objective To investigate the effect of human platelet-rich plasma-derived exosomes(PRP-exos)on the proliferation of Schwann cell(SC)cultured in vitro. Methods PRP-exos were extracted by polymerization-precipitation combined with ultracentrifugation.The morphology of PRP-exos was observed by transmission electron microscopy,and the concentration and particle size distribution of PRP-exos were determined by nanoparticle tracking analysis.Western blotting was employed to determine the expression of the marker proteins CD63,CD81,and CD9 on exosome surface and the platelet membrane glycoprotein CD41.The SCs of rats were isolated and cultured,and the expression of the SC marker S100β was detected by immunofluorescence staining.The fluorescently labeled PRP-exos were co-cultured with SCs in vitro for observation of their interaction.EdU assay was employed to detect the effect of PRP-exos on SC proliferation,and CCK-8 assay to detect the effects of PRP-exos at different concentrations(0,10,20,40,80,and 160 μg/ml)on SC proliferation. Results The extracted PRP-exos appeared as uniform saucer-shaped vesicles with the average particle size of(122.8±38.7)nm and the concentration of 3.5×1012 particles/ml.CD63,CD81,CD9,and CD41 were highly expressed on PRP-exos surface(P<0.001,P=0.025,P=0.004,and P=0.032).The isolated SCs expressed S100β,and PRP-exos could be taken up by SCs.PRP-exos of 40,80,and 160 μg/ml promoted the proliferation of SCs,and that of 40 μg/ml showed the best performance(all P<0.01). Conclusions High concentrations of PRP-exos can be extracted from PRP.PRP-exos can be taken up by SCs and promote the proliferation of SCs cultured in vitro.
目的 探究人富血小板血浆来源外泌体(PRP-exos)对体外培养的施万细胞(SC)增殖能力的影响。方法 采用聚合沉淀结合超速离心法提取PRP-exos,透射电镜观察其形态,纳米颗粒跟踪分析技术测定其浓度及粒径分布,Western blot检测外泌体表面标志蛋白CD63、CD81、CD9及血小板膜糖蛋白CD41。分离培养大鼠SC,免疫荧光染色检测SC标记分子S100β的表达。荧光标记PRP-exos,与SC体外共培养,观察两者的相互作用。EdU实验检测PRP-exos对SC增殖能力的影响,CCK-8法检测不同浓度(0、10、20、40、80、160 μg/ml)PRP-exos对SC增殖能力的影响。结果 提取的PRP-exos呈均匀的圆形囊泡,具有外泌体典型的茶托状形态,平均粒径(122.8±38.7)nm,浓度为3.5×1012个/ml,PRP-exos表面高表达CD63、CD81、CD9及CD41(P<0.001,P=0.025,P=0.004,P=0.032)。分离培养的SC表达S100β,PRP-exos可被SC摄取。浓度为40、80、160 μg/ml的PRP-exos可促进SC的增殖,且40 μg/ml PRP-exos的促增殖效果最佳(P均<0.01)。结论 PRP中可提取较高浓度的PRP-exos,体外培养条件下,PRP-exos能够被SC摄取,且对SC具有促增殖作用。.
Keywords: Schwann cell; exosomes; nerve regeneration; platelet-rich plasma; proliferation.