目的 探讨黄芪甲苷(AstragalosideⅣ,AS-Ⅳ)干预的人内皮祖细胞外泌体(Endothelial progenitor cells derived exosomes,EPC-Exos)对高糖诱导损伤人脐血间充质干细胞(Human umbilical cord blood mesenchymal stem cells,hUCBMSCs)向内皮分化的影响.方法 体外分离和培养人内皮祖细胞(Endothelial Progenitor Cells,EPCs),同时体外分离和培养hUCBMSCs,取P4代细胞分别进行成骨、成软骨和成脂诱导分化鉴定.将鉴定成功的EPCs分别用100mg·L-1的黄芪甲苷和等量的PBS干预,培养24 h后收集两组细胞上清液中外泌体.采用透射电子显微镜观察EPC-Exos的形态,利用纳米粒子追踪分析(Nanoparticle Tracking Analysis,NTA)技术检测EPC-Exos的粒径,Western blot技术进行外泌体特征性标志物CD9、CD63和TSG101的检测.将hUCBMSCs用30 mmol·L-1的葡萄糖预处理120h后,随机分为实验组和对照组,同时设置正常组.三组细胞培养24 h后,通过Matrigel体外成管实验研究EPC-Exos对hUCBMSCs成管分化的影响.免疫荧光检测hUCBMSCs表达CD31、vWF等内皮细胞特异标志物的情况.结果 光镜下hUCBMSCs边缘清楚,形态均一,排列呈漩涡状,3系细胞分化为典型图像.透射电镜观察分离及提纯后黄芪甲苷干预EPC-Exos为包膜完整的圆形或椭圆形微囊泡结构;NTA技术检测97.6％人EPC-Exos为直径在81.4-142.1nm之间的微囊泡;EPC-Exos表面特异标志物CD9、CD63、TSG101呈阳性,以上实验证实成功提取EPC-Exos.Matrigel成管实验显示,与对照组相比,实验组MSCs细胞体外成管能力显著增强,差异显著(P<0.001).免疫荧光检测显示,与对照组相比,实验组MSCs表达内皮细胞特异性标志物CD31、vWF能力显著增强,差异显著(P均<0.01).结论 黄芪甲苷干预的EPC-Exos可有效恢复高糖受损人间充质干细胞的成管功能且显著改善其向内皮分化能力.

目的 探讨人参皂苷Rg1(Ginsenoside-Rg1,G-Rg1)对内皮祖细胞(Endothelial progenitor cells,EPCs)分泌外泌体(Endothelial progenitor cells derived exosomes,EPC-Exos)及表达血管新生相关微小 RNA(MicroRNAs,miRNAs)的影响,为进一步研究G-Rg1介导的EPC-Exos促血管新生的作用机制奠定基础.方法 无菌条件下取正常剖宫产胎盘中的脐带血,分离得到单个核细胞,置于EGM-2培养基传代培养至P3代,通过CD31免疫磁珠分选法及双荧光染色法共同鉴定细胞.将鉴定成功的EPCs随机分为实验组及对照组,实验组用40mg·L-1G-Rg1干预,对照组用等体积PBS液处理,各培养24h.采用超速离心法结合超滤法提取EPC-Exos,通过透射电子显微镜观察其形态,蛋白印迹法鉴定其特征性表面标志物,BCA蛋白定量法检测总蛋白浓度,RT-qPCR法检测各组外泌体中与血管新生相关miRNAs分子的表达.结果 培养24h后,各组EPC-Exos表面标志物CD9、CD63和TSG101的表达均呈阳性.对照组与实验组EPC-Exos总蛋白浓度分别为(0.85±0.02)和(0.97±0.02)ug·uL-1,差异显著,有统计学意义(P＜0.01).实验组中部分促血管新生相关 miRNAs 表达较对照组增多,即 miRNA-126-5p、miRNA-146a-5p、miRNA-210、miRNA-214-5p 的表达明显增多,差异显著,有统计学意义(P＜0.05).结论 人参皂苷(G-Rg1)能促进内皮祖细胞分泌外泌体,使外泌体中与血管新生相关的miRNA-126-5p、miRNA-146a-5p、miRNA-210、miRNA-214-5p表达明显增多,可进一步从外泌体水平阐释G-Rg1促进血管新生的机制.

Introduction: The high glucose changes caused by diabetes mellitus (DM) can damage the vascular system. Astragaloside IV (AS-IV) can improve diabetes and promote angiogenesis. Exosomes (EXOs) help to carry specific drugs into cells efficiently. However, whether AS-IV loaded EXOs (AS-IV EXOs) can improve damaged endothelial cells through miR-214 remains to be determined. Material and methods: We prepared and identified AS-IV EXOs derived from endothelial progenitor cells (EPCs) and high glucose stimulated endothelial cell models to investigate whether AS-IV EXOs can improve damaged endothelial cells through miR-214. We used a transmission electron microscope (TEM) and DAPI staining to identify the morphology and characteristic expression of EPCs and EXOs, and then prepared AS-IV EXOs. Cell function tests were performed to detect the cloning, proliferation, and migration capabilities of cells. Western blot (WB) and real-time quantitative polymerase chain reaction (qRT-PCR) were used to assess the expression level of Tie-2, Ang-1, and PI3K/Akt-related protein. Results: The DAPI staining results showed that inducing human umbilical vein endothelial cells (HUVECs) could effectively absorb AS-IV EXOs. The results of plate clone formation assay, CCK-8, cell adhesion, and transwell assay of HUVECs stimulated by high glucose showed that AS-IV EXOs had a damage relief effect. By the detection of WB and qRT-PCR, it was found that AS-IV EXOs promoted the expression of miR-214 and proteins related to blood vessel growth. After transfection of miR-214 to pre-treat HUVECs under high glucose stimulation, AS-IV EXOs promoted the tube formation of HUVECs by regulating the level of miR-214. Conclusions: By promoting the expression of miR-214, AS-IV EXOs significantly improved the activity and tubularization of HUVECs under high glucose stimulation.

目的 观察人参皂苷Rg1对高糖环境下人脐带血间充质干细胞(hUCBMSCs)分泌炎症因子的影响.方法 无菌条件下取正常剖宫产胎盘中的脐带血,采用密度梯度离心法分离人脐带血间充质干细胞(hUCBMSCs)并鉴定.将鉴定成功的hUCBMSCs随机分为正常组、模型组、干预组.模型组和干预组加入含30 mmol/L葡萄糖的DMEM/F12培养基建立高糖受损细胞模型,正常组加入DMEM/F12培养基;培养5 d后,干预组加入40 mg/L的人参皂苷Rg1,正常组和模型组加入等量PBS,继续培养48 h.收集三组细胞上清液,采用酶联免疫吸附法检测炎症因子白细胞介素(IL)-1、IL-6、IL-10、巨噬细胞集落刺激因子(M-CSF)、单核细胞趋化蛋白1(MCP-1)水平.结果 与正常组相比,模型组细胞上清液中IL-1、IL-6、MCP-1升高(P均<0.05);与模型组相比,干预组细胞上清液中IL-1、IL-6降低,IL-10、M-CSF升高(P均<0.05).结论 高糖环境下,人参皂苷Rg1可抑制hUCBMSCs中的促炎因子释放,促进抑炎因子分泌,从而发挥抑制炎症反应的作用.

目的 探讨阿魏酸(ferulic acid,FA)对间充质干细胞(mesenchymal stem cells,MSCs)增殖、分泌干细胞因子(stem cell factor,SCF)和定向内皮细胞成管分化的影响.方法 取足月健康新生儿脐带血10 mL,采用密度梯度离心法得到单个细胞,并鉴定人脐带血间充质干细胞(human umbilical cord blood mesenchymal stem cells,hUCBMSCs)分化能力.将hUCBMSCs分别用不同浓度梯度的FA(0、1、2、4、8、16 mg/L)干预以确定FA促hUCBMSCs增殖的最佳浓度.取hUCBMSCs随机分为实验组与对照组,实验组用最佳浓度FA干预,对照组用等体积PBS液处理.分别采用CCK-8细胞增殖试验、Matrigel体外成管试验及ELISA法检测两组hUCBMSCs增殖、成管及分泌SCF的能力;采用免疫荧光法检测hUCBMSCs向内皮细胞分化后CD31、vWF的表达情况.结果(1)hUCBMSCs能成功诱导分化为骨细胞、软骨细胞、脂肪细胞;(2)FA促进hUCBMSCs增殖的最佳浓度为2 mg/L;(3)与对照组相比,实验组hUCBMSCs增殖、成管及分泌SCF能力显著增强,且FA诱导成管分化后CD31和vWF的表达明显增多(P<0.05).结论 FA能促进hUCBMSCs增殖及分泌SCF,同时能诱导hUCBMSCs成管分化,进一步证实FA具有促进血管新生的潜能.

Objective:To investigate the effect of astragaloside IV (AS-IV) on the secretion of stromal cell-derived factor-1α (SDF-1α) and CXC chemokine receptor 4 (CXCR4) by high glucose injured human umbilical vein endothelial cells (HUVECs), so as to lay a foundation for further study on AS-IV improving angiogenesis by regulating SDF-1 α/CXCR4 axis of endothelial cells.Methods:HUVECs were isolated and cultured from the umbilical vein of full-term healthy newborns and identified by von Willebrand factor (vWF) combined with 4-diamino-2-phenylindole (DAPI) nuclear staining. The obtained HUVECs was cultured in EGM-2 medium with 30 mmol/L glucose for 120 h to obtain high glucose damaged HUVECs. After intervention with different concentration gradients (25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L) AS-IV for 72 hours, the contents of SDF-1α and CXCR4 were detected by enzyme linked immunosorbent assay (ELISA) method to determine the best concentration of AS-IV. The supernatant of damaged HUVECs were collected at 6, 12, 24, 48 and 72 hours after intervention with the best concentration of AS-IV, and the contents of SDF-1α and CXCR4 were detected by ELISA method to determine the best action time of AS-IV. The damaged HUVECs was randomly divided into experimental group and control group, and the blank group was set up at the same time. The experimental group was treated with the best concentration of AS-IV and the best time, the control group and the blank group were treated with the same volume of phosphate buffered saline (PBS) solution, and the contents of SDF-1α and CXCR4 in each group were detected by ELISA method.Results:The vWF factor on the cell membrane was green fluorescence, and the nucleus was blue after DAPI staining. When the fusion image showed green fluorescence, HUVECs were identified by blue fluorescence. The expression of SDF-1α in damaged HUVECs was the best when treated with AS-IV of 100 mg/L for 24 hours (1 642.87 pg/ml), and the expression of CXCR4 in damaged HUVECs was the best when treated with AS-IV of 50 mg/L for 48 hours (8.44 ng/ml). Compared with the control group, the contents of SDF-1α and CXCR4 in the experimental group were significantly increased, and the difference was statistically significant ( P<0.05). While the contents of SDF-1α and CXCR4 in the experiment group were slightly less than those in the blank group and there was no statistically significant difference ( P>0.05). Conclusions:AS-IV can promote the expression of SDF-1α and CXCR4 in HUVECs damaged by high glucose to return to normal physiological level, so as to play the role of vascular repair and neovascularization.

Objective: To investigate the effects of Astragaloside Ⅳ on 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 Astragaloside Ⅳ group and phosphate buffer solution (PBS) group. The cells in Astragaloside Ⅳ group were cultured with Astragaloside Ⅳ in 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 Astragaloside Ⅳ group 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 Astragaloside Ⅳ group 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 Astragaloside Ⅳ group than in PBS group. Conclusions: Astragaloside Ⅳ can improve the function of human EPC secretory exosomes, and the secreted exosomes are loaded with miRNA-126.

目的:研究黄芪甲苷(astrologicalⅣ,AS-Ⅳ)对高糖受损人内皮祖细胞(endothelial progenitor cells,EPCs)分泌基质细胞衍生因子-1α(stromalcell-derived factor-1α,SDF-1α)和CXC趋化生长因子受体4(CXCchemokine receptor 4,CXCR4)的影响.方法:从足月健康新生儿脐带血中分离培养出单个核细胞,采用CD31抗体联合DAPI核染及FITC-UEA-I和Dil-ac-LDL双荧光染色法鉴定EPCs.用30 mmol/L葡萄糖预处理EPCs得到高糖受损EPCs.用不同浓度梯度(25、50、100、200、400 mg/L)的AS-Ⅳ干预EPCs,经ELISA法检测分别确定AS-Ⅳ促SDF-1α和CXCR4分泌的最佳浓度.用最佳浓度AS-Ⅳ干预受损的EPCs,分别于6、12、24、48、72h收集样本以确定AS-Ⅳ的最佳作用时间.将受损EPCs随机分为实验组和对照组,同时设置空白组.实验组用最佳浓度AS-Ⅳ干预,对照组及空白组用等容量的PBS液处理,用ELISA法检测各组SDF-1α和CXCR4的含量.结果:100 mg/L的AS-Ⅳ作用24 h时受损的EPCs分泌SDF-1α的量达最佳;50 mg/L的AS-Ⅳ作用48 h时受损的EPCs分泌CXCR4的量达最佳.与对照组比较,实验组EPCs分泌SDF-1α、CXCR4的含量明显增多,差异有统计学意义(P＜0.05).实验组EPCs分泌SDF-1α和CXCR4的含量与空白组比较,差异无统计学意义(P≥0.05).结论:AS-Ⅳ能促进高糖受损人EPCs分泌SDF-1α和CXCR4,且分泌量能达到正常生理状态.