Objective: We explore the effects of endothelial progenitor cell (EPC)-derived exosomes (EPCexos) and of astragaloside IV (ASIV)-stimulated EPCexos (ASIV-EPCexos) on type I diabetic-wound healing, and determine the basic molecular mechanisms of action. Methods: EPCs were exposed to different concentrations of ASIV to generate ASIV-EPCexos. A chronic-wound healing model involving streptozotocin-stimulated diabetic rats was established. These rats were treated with EPCexos, ASIV-EPCexos, rapamycin, and wortmannin. Wound healing was evaluated by direct photographic observation, hematoxylin and eosin staining, and Masson’s trichrome staining. Results: ASIV treatment increased the abilities of EPCs (e.g., proliferation), as well as exosome secretion. EPCexo showed a “cup holder” like structure. Treatment with ASIV-EPCexos increased the wound-healing rate, collagen-deposition area, bromodeoxyuridine uptake, VEGF expression, and the number of CD31- and αSMA- positive cells, whereas decreased epidermal thickness and CD45 expression. The expression of the PI3K/AKT/mTOR pathway increased, whereas the expression of inflammatory factor decreased. However, rapamycin and wortmannin reversed these changes. Conclusions: ASIV-EPCexos may accelerate type I diabetic-wound healing via the PI3K/AKT/mTOR pathway. This study may lay the foundation for new clinical treatment options for patients with type I diabetic wounds.

目的:探讨人参皂苷Rg1对高糖诱导损伤的内皮祖细胞(EPCs)生物学功能和分泌血管生成相关因子的影响.方法:体外分离和培养人脐带血EPCs,通过观察细胞形态、双荧光染色法对培养的EPCs进行鉴定.将鉴定成功的EPCs用30 mmol/L的葡萄糖预处理120 h后,分别在不同浓度梯度人参皂苷Rg1(0 mg/L、5 mg/L、10 mg/L、20 mg/L、40 mg/L、80 mg/L)条件下干预培养,确定人参皂苷Rg1促高糖受损EPCs增殖的最佳浓度.将高糖诱导损伤的EPCs随机分为实验组(最佳浓度人参皂苷Rg1干预)和模型组[磷酸缓冲盐溶液(PBS)干预],同时设置正常EPCs对照组(PBS干预).采用细胞计数试剂(CCK-8)、黏附能力测定试验、Matrigel体外成管试验、划痕实验及酶联免疫吸附试验(ELISA)法检测EPCs增殖、黏附、成管、迁移能力及EPCs分泌血管内皮生长因子(VEGF)、基质细胞衍生因子-1α(SDF-1α)、血管生成素-1(Ang-1)、基质金属蛋白酶-2(MMP-2)的水平.结果:人参皂苷Rg1促高糖受损EPCs增殖的最佳浓度为40 mg/L;与正常组比较,高糖受损EPCs增殖、黏附及成管能力明显降低(P<0.01);采用人参皂苷Rg1干预后,高糖受损EPCs增殖、黏附、成管及迁移能力明显增强(P<0.01).ELISA检测发现,与正常组比较,模型组EPCs分泌VEGF、SDF-1α、Ang-1明显减少,而MMP-2分泌增加,差异均有统计学意义(P<0.05);实验组在40 mg/L人参皂苷Rg1干预后,EPCs分泌VEGF、SDF-1α、Ang-1明显增加,而MMP-2分泌减少,差异有统计学意义(P<0.05).结论:人参皂苷Rg1处理能改善高糖受损EPCs的生物学功能及促进EPCs分泌血管生成相关生长因子,具有促血管新生的潜能.

目的 研究阿魏酸调控内皮祖细胞(endothelial progenitor cells,EPC)分泌外泌体(exosomes,Exo)及对表达血管新生微小RNA的影响.方法 将EPC随机分为实验组和对照组.实验组加入8 mg·L-1阿魏酸干预24 h,对照组用等容量的磷酸盐缓冲溶液干预24 h.用透射电子显微镜观察EPC-Exo形态,用蛋白质印迹法鉴定其特征性表面标志物,用二辛丁酸蛋白定量法检测总蛋白浓度,用实时荧光聚合酶链反应法检测EPC-Exo中血管新生相关miRNA分子的表达水平.结果 培养24 h后,各组EPC-Exo 呈圆形或椭圆形的膜囊泡结构,EPC-Exo特征性表面标志物CD9、CD63和TSG101的表达均呈阳性.干预24 h后,实验组与对照组的EPC-Exo蛋白浓度分别为(1.01±0.02)和(0.85±0.01)μg·μL-1,miR-21表达水平分别为 2.51±0.34 和 0.99±0.12,miR-126-5p 表达水平分别为3.97±0.45 和 1.04±0.03,miR-146a-5p 表达水平分别为8.04±0.52和1.06±0.05,miR-210表达水平分别为4.77±0.37和 1.12±0.03,miR-214-5p 表达水平分别为 0.74±0.06 和 0.39±0.02,差异均有统计学意义(均P＜0.05).结论 阿魏酸能促进EPC分泌Exo,且EPC-Exo中富含有与血管新生相关的miRNAs.

目的 探讨黄芪甲苷(AS-Ⅳ)对生信分析筛选出的人脐带血间充质干细胞(hUCBMSCs)外泌体(Exos)中与血管生成相关微小RNAs(miRNAs)表达的影响.方法 通过生信分析筛选hUCBMSCs-Exos中与血管生成高度相关的miRNAs.将hUCBMSCs随机分为实验组和对照组.实验组给予含300 mg·L-1 AS-Ⅳ的DMEM/F12培养基培养24 h,对照组加入等量磷酸盐缓冲溶液培养24 h.用二辛丁酸法检测Exos浓度,用实时荧光聚合酶链反应法检测miRNAs的表达水平.结果 实验组和对照组的外泌体浓度分别为(1.22±0.02)和(0.88±0.03)μg·μL-1,差异有统计学意义(P<0.01).miRNA-126-3p、miRNA-21、miRNA-214-5p、miRNA-126-5p、miRNA-145、miRNA-16-5p和miRNA-195-5p在实验组的表达量分别为6.69±0.57,1.06±0.16,1.06±0.21,0.68±0.05,0.51±0.17,0.48±0.14和0.46±0.10,在对照组中的表达量分别为1.04±0.14,0.74±0.10,0.48±0.14,1.04±0.14,1.02±0.17,1.09±0.15和1.18±0.20,差异均有统计学意义(均P<0.05).结论 AS-Ⅳ可改善hUCBMSCs分泌Exos的能力,且所分泌的Exos负载有与血管生成相关的miRNAs,具有强大的诱导血管新生的潜能.

目的 探讨人参皂苷 Rg-1(ginsenoside Rg1,GS-Rg1)对人内皮祖细胞(endothelial progenitor cells,EPCs)体外增殖和分泌肝细胞生长因子(hepatocyte growth factor,HGF)的影响,为进一步研究GS-Rg1通过EPCs介导血管新生的作用机制奠定基础.方法 取正常剖宫产胎盘中的脐带血,密度梯度离心分离出单个细胞;通过CD31免疫荧光鉴定和双荧光染色的共聚焦拍摄(DiI-acLDL和FITC-UEA-1配合DAPI染核)共同对EPCs进行鉴定;将鉴定成功的EPCs分别在不同浓度梯度Gs-Rg1(0、5、10、2、40、80 mg/L)下干预培养48 h,确定Gs-Rg1促EPCs增殖的最佳浓度.另将获得的EPCs随机分为实验组(最佳浓度Gs-Rg1干预)和对照组(PBS干预).采用细胞计数试剂盒(Cell Counting Kit-8,CCK-8)、流式细胞术及ELISA法分别检测GS-Rg1对EPCs的增殖、凋亡和分泌HGF的影响.结果 在一定范围(＜40 mg/L)内,GS-Rg1的浓度与EPCs增殖呈正相关,Gs-Rg1促EPCs增殖的最佳浓度为40 mg/L;与对照组相比,实验组细胞增殖、抗凋亡和分泌HGF的能力显著增强,差异显著,具有统计学意义(P＜0.05).结论 GS-Rg1对EPCs无毒性,且能改善其增殖功能和促进HGF的分泌.

目的 探讨黄芪甲苷(AS-Ⅳ)介导的内皮祖细胞外泌体(EPCs-Exos)对高糖受损内皮细胞(ECs)增殖和凋亡的影响.方法 从足月健康新生儿脐带血中分离、培养单个核细胞,用CD31抗体联合4,6-二脒基-2-苯基吲哚(DAPI)核染鉴定,获得的EPCs用100 mg·L-1 AS-Ⅳ干预24 h.用超速离心法结合超滤法提取细胞上清液中囊泡结构,用透射电子显微镜进行形态观察,用蛋白质印迹(WB)法检测外泌体表面特征性标志蛋白CD9、CD63、CD81.体外分离、培养人脐静脉内皮细胞(HUVECs),用vWF因子联合DAPI核染鉴定,获得的HUVECs用30 mmol·L-1葡萄糖干预120 h,随机分为实验组、对照组并设置正常组(用正常EGM-2培养基预培养120 h).实验组用AS-IV介导下的EPCs-Exos处理24 h,对照组和正常组用等容量PBS处理24 h,用CCK-8细胞增殖检测试剂盒,用流式细胞术分别检测细胞增殖和凋亡情况.结果 正常组、对照组和实验组OD值分别为0.73±0.00,0.45±0.00和0.74±0.01,凋亡率分别为(6.44±0.27)％,(24.41±1.12)％和(9.54±0.20)％.各组细胞凋亡率比较,差异均有统计学意义(均P<0.05).结论 高糖环境下ECs增殖能力抑制明显、凋亡率上升,而AS-IV介导的EPCs-Exos可显著促进高糖受损ECs增殖并抑制其凋亡,对ECs有明确的保护作用.

Objective:To investigate the impacts of astragaloside Ⅳ (AS-Ⅳ) on in- vitro proliferation and angioblastic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUCBMSCs), providing a basis for further research about the effects of AS-Ⅳ on mesenchymal stem cells (MSCs)-mediated angiogenesis. Methods:The hUCBMSCs were extracted from umbilical cord blood of normal full-term infants and subcultured. Osteoblasts, chondroblasts, and lipoblasts were induced, differentiated and identified. At the same time, the surface antigens CD44, CD73, and CD105 on hUCBMSCs were determined by flow cytometry. The successfully identified hUCBMSCs were cultured and treated with a series concentrations of AS-Ⅳ (0, 50, 100, 200, 300, and 400 mg/L). The optimum concentration of AS-Ⅳ for cell proliferation in hUCBMSCs was confirmed. In another experiment, hUCBMSCs were randomly divided into the experimental group and the control group. The cells in the experiment group were treated with the optimum concentration of AS-Ⅳ, and those in the control group were treated with equal volume of PBS. The impact of AS-Ⅳ on the proliferation of hUCBMSCs was detected using the cell counting kit (CCK-8). Besides, the impact of AS-Ⅳ on the angioblastic differentiation of hUCBMSCs was examined using the matrigel in- vitro tube formation assay. CD31 and von willebrand factor (vWF) expressions were determined using immunofluorescence after hUCBMSCs differentiated towards endothelial cells. Results:Under the light microscope, hUCBMSCs had clear edges and arranged orderly, showing a typical long fusiform structure. Flow cytometry confirmed that hUCBMSCs had surface markers of mesenchymal stem cells. The optimum concentration of AS-Ⅳ for the proliferation of MSCs was 300 mg/L. The OD values of the control and experimental groups were (0.51±0.01) and (0.98±0.05), respectively, with statistical significance ( t=15.96, P<0.05), indicating that the proliferation ability of the experimental group was enhanced. Compared with the control group, the tube density and the length of the tube network in vitro in the experimental group were higher, with statistically significant difference [(629.80±52.94)mm vs (110.36±13.19)mm, P<0.05]. Compared with the control group, the expression of CD31 and vWF increased in the experimental group after AS-Ⅳ induced hUCBMSCs differentiation ( t=13.64, 13.18, P<0.05). Conclusions:AS-Ⅳ has no toxicity to human umbilical cord blood mesenchymal stem cells, and can improve their proliferation function, and induce hUCBMSCs to differentiate into endothelial cells.

目的 探讨阿魏酸(FA)对高糖诱导损伤的内皮祖细胞(EPCs)生物学功能和超氧化物歧化酶(SOD)的影响.方法 体外分离和培养人脐带血EPCs,通过观察细胞形态、双荧光染色法及免疫荧光细胞化学染色法等技术对培养的EPCs进行鉴定.将鉴定成功的EPCs用30 mmol/L的葡萄糖预处理120 h后,分别在不同浓度梯度FA(0、1、2、4、8、16 mg/L)条件下干预培养,确定FA促高糖受损EPCs增殖的最佳浓度.另将高糖诱导损伤的EPCs随机分为实验组(最佳浓度FA干预)和模型对照组(PBS干预),同时设置正常EPCs对照组(PBS干预).采用细胞计数试剂盒(CCK-8)、黏附能力测定试验、Matrigel体外成管试验、划痕实验及ELISA法检测FA对细胞增殖、黏附、成血管、迁移能力及对EPCs分泌SOD的影响.结果 FA促高糖受损EPCs增殖的最佳浓度为8 mg/L;与正常组比较,高糖受损EPCs增殖、迁移、黏附及成管能力显著降低(P＜0.05);当用FA干预后,高糖受损EPCs增殖、黏附、迁移及成血管能力显著增强(P＜0.05).ELISA检测发现,与正常组比较,高糖受损EPCs分泌SOD能力显著下降(P＜0.05),而用FA干预后,能部分逆转EPCs分泌SOD能力,但差异无统计学意义(P＞0.05).结论 FA处理能改善高糖受损EPCs生物学功能,但对高糖受损EPCs分泌SOD作用不明显.

目的 观察研究人参皂苷Rg1(GS-Rg1)对高糖诱导损伤人脐血间充质干细胞(hUCBMSCs)生物学特性及分泌干细胞因子(SCF)的影响.方法 通过体外分离和培养hUCBMSCs,并取第4代细胞进行成骨、成软骨、成脂诱导分化鉴定.将鉴定成功的hUCBMSCs用含30 mmol/L葡萄糖的培养基培养120 h,分别用不同浓度GS-Rg1(0、5、10、20、40、80 mg/L)处理,确定GS-Rg1促进高糖诱导损伤hUCBMSCs增殖的最佳浓度.将高糖诱导损伤hUCBMSCs分成试验组和对照组,另将用含5.5 mmol/L葡萄糖的培养基培养的hUCBMSCs作为正常组.试验组用最佳浓度GS-Rg1干预,对照组和正常组用等容积磷酸盐缓冲液(PBS)干预,分别观察各组对高糖诱导损伤hUCBMSCs的增殖能力、黏附能力、迁移能力、抗凋亡能力及分泌SCF功能的影响.结果 GS-Rg1促进高糖诱导损伤hUCBMSCs增殖的最佳浓度为40 mg/L.与正常组比较,对照组hUCBMSCs的增殖密度(OD)值、黏附数、迁移宽度及SCF含量均明显下降(P<0.05),细胞凋亡率明显升高(P<0.05);与对照组比较,试验组hUCBMSCs增殖OD值、黏附数、迁移宽度及SCF含量均明显增加(P<0.05),细胞凋亡率明显下降(P<0.05).结论 GS-Rg1对高糖诱导损伤hUCBMSCs的生物学特性具有保护作用,能够明显促进hUCBMSCs增殖,提高黏附和迁移能力,减少细胞凋亡,并能促进SCF分泌.

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-mediated Endothelial progenitor cells derived exosomes (EPC-Exos) on the biological function of EPC-Exos damaged by high glicose.Methods:EPCs from human umbilical cord blood were isolated and cultured in vitro. the EPC-Exos secreted by EPCs were extracted by ultracentrifugation combined with ultrafiltration, and identified by specific markers CD9, CD63 and CD81, respectively. After the cells were cultured for 24 hours with AS-IV at 100 mg/L and PBS at the same volume, the morphological characteristics of EPC-Exos were observed by transmission electron microscope. Human endothelial cells were isolated, cultured and identified in vitro. The identified endothelial cells were pretreated with 30 mmol/L glucose for 120 h and randomly divided into experimental group and control group, at the same time set the normal group. The cells were cultured for 24 hours, the effects of EPC-Exos on proliferation, adhesion, migration and angiogenesis of endothelial cells damaged by high glucose were observed by using cell counting kit-8 (CCK-8) Cell Proliferation Assay Kit, cell scratch test, adhesion assay and in vitro angiogenesis assay by Matrigel. Results:Compared with the normal group, the proliferation, migration, adhesion and tubulogenesis of human endothelial cells in the control group were significantly lower ( t=24.35, 6.80, 10.65, 9.62, P<0.05). Compared with the control group, the proliferation, adhesion, migration and tubulogenesis of human endothelial cells in the experimental group were significantly enhanced ( t=30.68, 5.99, 5.40, 8.25, P<0.05). Conclusions:EPC-Exos mediated by AS-Ⅳ can significantly improve the biological function of human endothelial cells damaged by high glucose and has the potential to modulate endothelial neovascularization in diabetic rats.

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.

目的:研究黄芪甲苷(Astragaloside IV,AS-IV)干预高糖受损人内皮祖细胞(Endothelial Progenitor Cells,EPCs)增殖的最佳浓度,并探讨AS-IV对高糖受损EPCs生物学功能的影响.方法:取足月新生儿脐带血分离、培养并鉴定EPCs,将鉴定成功的EPCs用30 mmol/L的葡萄糖预处理120 h后,随机分为实验组(HG+AS-IV组)和对照组(HG组),同时设置正常组(NC组).用CCK8检测不同浓度AS-IV(0、25、50、100、200、400 mg/L)干预EPCs 24 h后的增殖情况,绘制增殖曲线,初步得出AS-IV干预高糖受损人EPCs增殖的最佳浓度.进而通过CCK-8增殖实验、黏附能力测定试验、细胞划痕试验及Matrigel体外成血管实验检测最佳浓度的AS-IV对高糖受损EPCs增殖、黏附、迁移和成管功能的影响.结果:AS-IV促高糖受损EPCs增殖的最佳浓度为100 mg/L;与对照组比较,100 mg/L AS-IV干预后的高糖受损EPCs增殖能力、黏附细胞数、细胞迁移率、体外成管数均明显增加,差异均有统计学意义(P1<0.05);同时检测实验组与正常组差异无统计学意义(P2>0.05).结论:AS-IV可显著改善体外高糖受损的人EPCs的生物学功能,恢复其原有活力并具有介导血管新生的潜能.