黄芪甲苷促进高糖受损的内皮祖细胞向内皮细胞分化的实验研究

Experimental Study on Differentiation of High Glucose Damaged Endothelial Progenitor Cells into Endothelial Cells Facilitated by AstragalosideⅣ

目的:研究黄芪甲苷(AS-Ⅳ)诱导高糖损伤内皮祖细胞(EPCs)向内皮细胞(ECs)分化的作用。方法:体外分离出单个核细胞,通过4,6二脒基-2-苯基吲哚(DAPI)核染法、FITC-UEA-I联合Dil-ac-LDL双荧光染色法鉴定EPCs,将鉴定成功的EPCs用30 mmol/L的葡萄糖干预120 h制成高糖受损模型,并随机分为高糖对照组及高糖AS-Ⅳ组,同时设置正常组。高糖AS-Ⅳ组加入100 mg/L的AS-Ⅳ干预,高糖对照组及正常组加入同等体积的PBS液培养。3组均培养48 h后采用荧光免疫检测内皮细胞标志物血小板-内皮细胞黏附分子(CD31)、血管性血友病因子(vWF)的表达情况,并通过Matrigel成管实验研究AS-Ⅳ干预EPCs成管分化的影响。结果:成功培养鉴定出EPCs,EPCs在光镜下形态呈中间圆形、外周梭形的“铺路石”样形态;双荧光染色荧光镜下EPCs呈双染橙黄色改变;免疫荧光检测显示,高糖AS-Ⅳ组CD31和vWF免疫荧光强度高于高糖对照组,差异有统计学意义(P<0.01);Matrigel成管实验显示,高糖AS-Ⅳ组EPCs成管能力高于高糖对照组,差异有统计学意义(P<0.01)。结论:AS-Ⅳ可诱导高糖受损人内皮祖细胞向内皮细胞分化而发挥体外成管作用。

Objective:To examine the differentiation of endothelial progenitor cells(EPCs)into endothelial cells(ECs)facilitated by AstragalosideⅣ(AS-Ⅳ).Methods:Mononuclear cells were isolated in vitro,with EPCs being identified through DAPI nuclear staining and dual fluorescence staining using FITC-UEA-I and Dil-ac-LDL.EPCs that were successfully identified underwent a 120-hour exposure to 30 mmol/L glucose to establish a model of high-glucose impairment.These were then allocated into high-glucose group,and high-glucose AS-Ⅳtreatment group,and control group was set up.The AS-Ⅳgroup received a 100 mg/L dosage of AS-Ⅳ,while the high-glucose group and control group were treated with an equivalent volume of PBS solution.After 48 hours of incubation,the expressions of endothelial markers CD31 and vWF were assessed using fluorescence immunoassays.The influence of AS-Ⅳon EPCs differentiation was further explored through Matrigel tube formation assays.Results:EPCs were successfully cultivated and identified,with a cobblestone appearance with rounded centers and spiky peripheries under light microscopy,and dual fluorescence staining revealed an orange-yellow hue.The AS-Ⅳgroup showed higher immunofluorescence intensity of CD31 and vWF than high-glucose group,with statistical significance(P<0.01).The Matrigel assay highlighted a marked improvement in the tube formation capabilities of EPCs within the AS-Ⅳgroup in contrast to the high-glucose group(P<0.01).Conclusion:AS-Ⅳhas the potential to induce the differentiation of human EPCs into ECs and enhance tube formation in vitro under conditions of high-glucose-induced damage.