目的 研究人参Panax ginseng根外泌体提取、表征及其体外心肌保护活性。方法 采用差速离心法与蔗糖梯度离心法提取人参根外泌体；通过透射电子显微镜（TEM）和纳米颗粒跟踪分析（nanoparticle tracking analysis，NTA）技术对外泌体进行鉴定；采用液相色谱-质谱联用（LC-MS）法对人参根外泌体进行蛋白质组学分析；通过Small RNA测序分析人参根外泌体中Small RNA种类；建立H9C2心肌细胞损伤模型，利用CCK-8法检测细胞活力；以最佳质量浓度的人参根外泌体处理H9C2细胞，应用Hoechst染色和JC-1染色考察细胞凋亡率；蛋白质免疫印迹法检测细胞凋亡相关蛋白。结果TEM图中清晰可见人参根外泌体呈茶托状的双层膜结构；NTA粒径分析检测到112.7 nm的人参根外泌体占总人参根外泌体的92.7%；蛋白组学分析鉴定到5585个蛋白；Small RNA测序鉴定到miRNA、tRNA、snRNA、rRNA、snoRNA 5种Small RNA；体外实验结果显示，与对照组相比，模型组细胞活力下降，人参根外泌体给药组呈剂量依赖性地使细胞活力升高，细胞凋亡率降低；Western blotting结果显示凋亡相关蛋白Bcl-2蛋白水平下降；Bax、Cyt C的蛋白水平升高。结论 首次报道了人参根外泌体的提取工艺及表征，并发现人参根外泌体对多柔比星诱导的心肌损伤具有一定的保护作用。

Objective To study the extraction process of ginseng root exosomes and its cardiac protective activity. Methods Extraction of ginseng root exosomes was carried out by differential centrifugation and sucrose gradient centrifugation; The exosomes were identified by transmission electron microscope and NTA granulometric analysis; Proteomic analysis of ginseng root exosomes was performed by liquid chromatography-mass spectrometry; Small RNA species in ginseng root exosomes were analyzed by Small RNA sequencing; H9C2 cardiomyocytes injury model was established and the cell viability was detected by CCK-8 assay; H9C2 cells were treated with the best concentration of ginseng root exosomes, and the apoptosis rate was determined by Hoechst and JC-1 staining; The apoptosis of related proteins was detected by Western blot. Results Double-layer membrane structure of ginseng root exosomes was clearly visible under transmission electron microscope; NTA particle size analysis showed that ginseng root exosomes with 112.7 nm particle size length was accounting for 92.7% of the total ginseng root exosomes. A total of 5585 proteins were identified by proteomic analysis; Small RNA sequencing identified five Small RNAs: miRNA, tRNA, snRNA, rRNA and snoRNA. The results of in vitro experiments showed that the cell viability of the model group was lower than that of the control group. Ginseng root exosomes increased cell viability and decreased apoptosis rate in a dose-dependent manner; The results of Western Blot showed that Bcl-2 protein level was decreased. Bax, Cyt C protein levels were increased. Conclusion In this study, the extraction process and identification of ginseng root exosomes were reported for the first time, and the protective effect of ginseng root exosomes on doxorubicin-induced myocardial injury was found.

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国家自然科学基金资助项目（81973712）；国家自然科学基金资助项目（81803680）；国家自然科学基金资助项目（82003985）；中国博士后科学基金资助项目（2020M670825）；中国博士后科学基金资助项目（2020T130568）