目的 探索根茎类中药甘草鲜药材来源的细胞外囊泡（Glycyrrhiza uralensis derived exosome-like nanovesicles，GELNs）的提取工艺和性质表征，并考察其抑制乳腺癌细胞增殖及血管新生作用。方法 根据根茎类中药材性质，优选药材处理方法和提取工艺，采用动态光散射法、透射电子显微镜、纳米流式、脂质组学、蛋白质组学等方法对提取所得GELNs进行理化性质表征和物质组成分析；从细胞摄取、细胞增殖和细胞凋亡角度，考察GELNs对小鼠乳腺癌4T1细胞的体外抗肿瘤作用；采用人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVEC）成管实验，探究GELNs抑制血管生成作用。结果 采用酶解法结合差速离心法提取得到GELNs，纳米颗粒跟踪分析（nanoparticle tracking analysis，NTA）、纳米流式测定粒径分布分别为（79.00±25.60）、（69.80±19.70）nm，其磷脂双分子膜结构占比75.20%；通过蛋白质组学与脂质组学分析，脂质组学共鉴定29种脂质亚类，其中三酰甘油占比最高，蛋白质组学定量38种蛋白并进行相关功能预测；4T1、HUVEC细胞均可高效摄取GELNs，其摄取结果呈剂量相关性，在细胞内主要分布于细胞质部位；GELNs可有效抑制4T1细胞增殖，且诱导4T1细胞发生程序性凋亡；GELNs可有效抑制HUVECs的体外成管，且呈剂量相关性。结论 对GELNs提取工艺和结构组成进行了研究，初步考察了其在4T1、HUVEC细胞的摄取能力、细胞增殖影响、促细胞凋亡和影响血管形成能力，提示GELNs是一种具有抗乳腺癌潜力的天然来源活性物质。

Objective To explore the extraction process and characterization of rhizome Chinese medicine Glycyrrhiza uralensis derived exosome-like nanovesicles (GELNs), and investigate their inhibitory effects on breast cancer cell proliferation and angiogenesis. Methods Based on the properties of rhizome-type traditional Chinese medicinal materials, the preferred processing methods and extraction techniques were selected, GELNs were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), nano-flow cytometry (NanoFCM), lipidomics and proteomics to analyze physicochemical properties and composition. The antitumor effects of GELNs against mouse breast cancer 4T1 cells were assessed through cellular uptake, proliferation inhibition and apoptosis induction. Human umbilical vein endothelial cells (HUVEC) tube formation assays was used to evaluate anti-angiogenic activity. Results GELNs were extracted by enzymatic hydrolysis method and differential centrifugation. Nanoparticle tracking analysis (NTA) and NanoFCM were used to determine the particle size distribution, which were (79.00 ± 25.60) and (69.80 ± 19.70) nm, respectively. The proportion of phospholipid bilayer membrane structure was 75.20%; Proteins and lipids in GELNs were identified and quantified, Lipidomics identified a total of 29 lipid class, among which triglycerides accounted for the highest proportion. Proteomics quantified 38 proteins and conducted relevant functional predictions; 4T1 and HUVEC cells could efficiently uptake GELNs, and the uptake results were dose-dependent, mainly distributed in the cytoplasm of cells; GELNs could effectively inhibit the proliferation of 4T1 cells and induce programmed apoptosis of 4T1 cells; GELNs could effectively inhibit the in vitro tube formation of HUVECs and exhibit a dose-dependent effect. Conclusion The extraction process and structural composition of GELNs were studied, and their uptake ability in 4T1 and HUVEC cells, effects of cell proliferation, apoptosis promotion and angiogenesis were preliminarily investigated. It is suggested that GELNs are a natural source active substance with anti-breast cancer potential.

R283；R285.5

校院联合创新基金项目（LH202402003）