黄芪甲苷对人内皮祖细胞分泌外泌体及表达微小RNA-126的影响

Effects of AstragalosideⅣon exosome secretion and its microRNA-126 expression in human endothelial progenitor cells

目的探讨黄芪甲苷对人内皮祖细胞(EPC)分泌外泌体及外泌体中微小RNA-126(miRNA-126)表达的影响。方法取湖南中医药大学第一附属医院妇产科2019年出生的1名足月健康新生儿脐带血,采用密度梯度离心法分离单个核细胞并培养7 d,其间行形态学观察;取第3代细胞,采用CD31免疫磁珠分选法和双荧光染色法鉴定。将鉴定成功的EPC按照随机数字表法分为黄芪甲苷组和磷酸盐缓冲液(PBS)组。黄芪甲苷组细胞加入终质量浓度100 mg/L的黄芪甲苷培养24 h,PBS组细胞加入等体积的PBS培养24 h。培养结束后收集2组细胞培养上清液中的外泌体,采用蛋白质印迹法检测外泌体特征性标志物CD9、CD63和CD81表达,于透射电子显微镜下观察EPC外泌体(EPC-Exo)形态,采用纳米颗粒跟踪分析技术检测EPC-Exo的粒径,采用二辛丁酸法测定EPC-Exo的浓度(样本数为3),采用反转录PCR法测定EPC-Exo中与血管新生相关miRNA-126-3p和miRNA-126-5p的表达(样本数为3)。对数据行独立样本t检验。结果(1)培养第4天,细胞开始贴壁生长,圆形、梭形、条形等多形态同时出现;继续培养至第7天时,细胞边缘清晰,呈铺路石样排列,中央细胞呈圆形,周边细胞呈梭形。(2)CD31免疫磁珠分选法显示细胞膜染绿色,细胞核染蓝色;双荧光染色法显示细胞呈橙黄色。经以上鉴定,细胞被证实为EPC。(3)培养24 h,2组EPC-Exo的CD9、CD63和CD81表达均呈阳性,证实本实验已成功提取EPC-Exo。(4)培养24 h,2组EPC-Exo均呈圆形膜囊泡,形态无明显差异。(5)培养24 h,黄芪甲苷组中98.7%的EPC-Exo粒径为84.7~143.1 nm,PBS组中98.0%的EPC-Exo粒径为88.7~123.5 nm。(6)培养24 h,黄芪甲苷组EPC-Exo的质量浓度为(310±5)μg/mL,明显高于PBS组的(257±5)μg/mL,t=13.369,P<0.01。(7)培养24 h,黄芪甲苷组EPC-Exo中miRNA-126-3p(t=16.062,P<0.01)和miRNA-126-5p(t=3.252,P<0.05)均明显多于PBS组。结论黄芪甲苷可改善人EPC分泌外泌体的功能,且所分泌的外泌体负载miRNA-126。

Objective To investigate the effects of AstragalosideⅣon the secretion of exosomes in human endothelial progenitor cells(EPCs)and the expression of microRNA(miRNA)-126 in exosomes.Methods The umbilical cord blood from one healthy full-term newborn from the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine in 2019 was harvested for isolating mononuclear cells by density gradient centrifugation and cultured for 7 days.Morphological observation was performed during this period.Cells of the third passage were collected for identification by CD31 immunomagnetic bead sorting and double fluorescence staining.According to the random number table,the identified EPCs were divided into AstragalosideⅣgroup and phosphate buffer solution(PBS)group.The cells in AstragalosideⅣgroup were cultured with AstragalosideⅣin final mass concentration of 100 mg/L for 24 hours,and the cells in PBS group were cultured with the same volume of PBS for 24 hours.After culture,the exosomes from the cell culture supernatant of the two groups were collected,and the expressions of characteristic markers of exosomes CD9,CD63,and CD81 were detected by Western blotting,the morphology of EPC exosomes(EPC-Exos)was observed under transmission electron microscope,and the particle size of EPC-Exos was detected by nanoparticle tracking analysis technique.The concentration of EPC-Exos was determined by dioctyl butyric acid method(the sample number was 3),and the expressions of miRNA-126-3p and miRNA-126-5p related to angiogenesis in EPC-Exos were determined by reverse transcription polymerase chain reaction(the sample number was 3).Data were statistically analyzed with independent sample t test.Results(1)On the 4th day of culture,the cells began to adhere to the wall,and the multi-forms such as circle,fusiform,and strip appeared at the same time.On the 7th day of culture,the edge of the cells was clear and arranged like a paving stone,the central cells were round,and the surrounding cells were fusiform.(2)CD31 immunomagnetic beads sorting method identification showed that the membrane was stained with green fluorescence and the nucleus was stained with blue fluorescence.Double fluorescence staining method showed that the cells were orange-yellow.The cells were identified as EPCs.(3)After 24 hours of culture,the expressions of CD9,CD63,and CD81 in EPC-Exos were all positive,confirming that EPC-Exos were extracted successfully in this experiment.(4)After 24 hours of culture,the EPC-Exos of the two groups showed round membrane vesicles,and there was no significant difference in morphology.(5)After 24 hours of culture,the particle size of 98.7%EPC-Exos in AstragalosideⅣgroup was 84.7 to 143.1 nm,and that of 98.0%EPC-Exos in PBS group was 88.7 to 123.5 nm.(6)After 24 hours of culture,the mass concentration of EPC-Exos in AstragalosideⅣgroup was(310±5)μg/mL,which was significantly higher than(257±5)μg/mL in PBS group,t=13.369,P<0.01.(7)After 24 hours of culture,there were more miRNA-126-3p(t=16.062,P<0.01)and miRNA-126-5p(t=3.252,P<0.05)in EPC-Exos of AstragalosideⅣgroup than in PBS group.Conclusions AstragalosideⅣcan improve the function of human EPC secretory exosomes,and the secreted exosomes are loaded with miRNA-126.