超速离心法和尺寸排阻色谱法对干细胞外泌体促血管生成及骨再生的影响

Effect comparison of stem cell-derived exosomes on angiogenesis and osteogenesis obtained by ultracentrifugation versus size-exclusion chromatography

目的:比较超速离心法和尺寸排阻色谱法提取干细胞外泌体的产率、纯度、生物活性和促骨修复能力的差异。方法:采用超速离心法和尺寸排阻色谱法提取骨髓间充质干细胞外泌体,对外泌体的粒径、外观形貌和标志蛋白进行鉴定表征。采用二喹啉甲酸(BCA)蛋白定量试剂盒测定外泌体中蛋白含量,并采用血管生成因子芯片对外泌体中成血管相关蛋白进行分析。采用划痕实验和小管形成实验评价外泌体体外促血管生成能力,进一步在大鼠骨缺损模型中评价外泌体对骨修复再生的效果。结果:单位体积上清液中,超速离心法所得外泌体(Exo-UC)质量低于尺寸排阻色谱法所得外泌体(Exo-SEC)质量。电镜下见Exo-SEC中杂质蛋白少于Exo-UC中的杂质蛋白。Exo-UC中检测到16种成血管蛋白,Exo-SEC中检测到的成血管蛋白种类和含量均少于Exo-UC。与Exo-SEC比较,Exo-UC能够显著促进血管内皮细胞迁移和小管形成,并显著提升大鼠骨缺损部位的骨体积百分比和骨矿物质密度。结论:超速离心法提取的干细胞外泌体促新生血管形成能力更强,骨修复效果更佳。

Objective:To compare the yield,purity,bioactivity,and bone repair potential of stem cell-derived exosomes isolated using ultracentrifugation and size-exclusion chromatography.Methods:Exosomes were isolated from the supernatant of bone marrow mesenchymal stem cells via ultracentrifugation and size-exclusion chromatography.The exosomes were characterized by evaluating their particle size,morphology,and marker proteins.The protein content in the exosomes was quantified via a bicinchoninic acid(BCA)method,and angiogenesis-related proteins were analyzed via an angiogenesis factor array kit.In vitro angiogenic potential was assessed through scratch and tube formation assays.Additionally,the ability of exosomes to promote bone repair and regeneration was evaluated in a rat bone defect model.Results:The mass of exosomes obtained via ultracentrifugation(Exo-UC)per volume of supernatant was lower than those obtained via size-exclusion chromatography(Exo-SEC).Transmission electron microscopy revealed fewer impurity proteins in Exo-SEC than in Exo-UC.Exo-UC contained 16 angiogenic proteins,whereas Exo-SEC contained fewer angiogenic proteins in terms of both species and content.Compared with Exo-SEC,Exo-UC significantly enhanced endothelial cell migration and tube formation.Furthermore,Exo-UC significantly increased the bone volume percentage and bone mineral density at bone defect sites in rats.Conclusions:Exosomes extracted from stem cells via ultracentrifugation demonstrate enhanced angiogenic potential and superior bone repair efficacy.