外源性Ang-1与miR-210共感染的内皮祖细胞腔内移植促进糖尿病慢性下肢缺血血管新生OA
Endovascular transplantation of endothelial progenitor cells co-infected with exogenous Ang-1 and miR-210 promotes angiogenesis in diabetic chronic lower limb ischemia
目的 探讨血管生成素-1(Ang-1)与微小RNA-210(miR-210)共修饰内皮祖细胞(EPCs)的抗凋亡、迁移及促血管新生能力,为糖尿病性慢性下肢缺血治疗提供实验依据.方法 分离SD大鼠骨髓EPCs,通过Dil-ac-LDL/FITC-UEA-1 双荧光染色及流式细胞测量术(CD34+/CD133+)鉴定表型.构建Ang-1 和miR-210 过表达慢病毒载体,并通过慢病毒感染EPCs,RT-qPCR检测基因表达.在高糖低氧(22.0mmol/L 葡萄糖,3%O2)条件下,采用Annexin V/PI 染色、Transwell小室法及ECMatrix胶血管生成实验,分别评估细胞凋亡率、迁移能力及成管效率.建立大鼠后肢缺血模型,腔内移植修饰 EPCs 后 28 d,行 CD31 免疫组化检测微血管密度,Western blot 分析Ang-1/Tie2/PI3K/AKT通路相关蛋白质表达.结果 成功培养鉴定 EPCs,共感染组 Ang-1 和 miR-210 表达量分别较对照组升高 3.88 倍和 4.21 倍(P＜0.01).高糖低氧环境下,共感染组EPCs凋亡率(10.84%)显著低于对照组(26.22%,P＜0.01),迁移细胞数(78.3±5.2)提高 3.64 倍(P＜0.01),血管分支数(15.6±1.8)增加3.7 倍(P＜0.01).动物实验显示,共感染组微血管密度(97.7±12.5)较对照组(26.3±18.4)提高 3.71倍(P＜0.05),缺血组织中 Ang-1、VEGF、p-AKT 及 p-VEGFR 表达显著上调(P＜0.05).结论 Ang-1 与miR-210 共修饰可显著增强 EPCs 在糖尿病缺血微环境中的存活能力与促血管新生功能,其机制与激活Ang-1/Tie2/PI3K/AKT通路密切相关.
Objective To investigate the co-modification of endothelial progenitor cells(EPCs)by angiopoietin-1(Ang-1)and microRNA-210(miR-210).The anti-apoptosis,migration and angiogenesis promotion of endothelial progenitor cells(EPCs)provide experimental basis for the treatment of diabetic chronic lower limb ischemia.Methods EPCs in bone marrow of SD rats were isolated and phenotype was identified by Dil-ac-LDL/FITC-UEA-1 double fluorescence staining microscopy and by flow cytometry(for checking CD34+/CD133+).Ang-1 and miR-210 over-expressed lentiviral vectors were constructed and infected into EPCs,gene expression was de-tected by RT-qPCR.Annexin V/PI staining,Transwell assay and ECMatrix gel angiogenesis assay were used to evaluate cell apoptosis rate,migration and tube formation in medium with high glucose and low oxygen(22.0 mmol/L glucose,3%O2)conditions.The rat hind limb ischemia model was established;And 28 days af-ter intracavity transplantation modified EPCs;Micro-vascular density was detected by CD31 immuno-histochemis-try;And Ang-1/Tie2/PI3K/AKT path-related protein expression was analyzed by Western blot.Results EPCs was successfully cultured and identified.The expression of Ang-1 and miR-210 in the co-transfection group was 3.88 times and 4.21 times higher than those in the control group respectively(P＜0.01).The EPCs apoptosis rate(10.84%)in the co-transfection group was significantly lower than that in the control group(26.22%,P＜0.01).The counting of migrating cells(78.3±5.2)was increased by 3.64 times(P＜0.01).The quantity of vascular branches(15.6±1.8)was increased by 3.7 times(P＜0.01).The microvascular density(97.7±12.5)in the cotransfection group was 3.71 times higher than that in the control group(26.3±18.4,P＜0.05)and the expression of Ang-1,VEGF,P-Akt and P-VEGFR in ischemic tissue was significantly up-regulated(P＜0.05).Conclusions The co-modification of Ang-1 and miR-210 can significantly improve the survival of EPCs in diabetic ischemic micro-environment and promote angiogenesis.Its mechanism is closely related to the activation of Ang-1/Tie2/PI3K/AKT pathway.
李春孟;孙慧艳;郑祥坚;谢尚尚;林德永;刘子田
温州市中心医院(温州医科大学 定理临床学院)血管外科,浙江 温州 325000||温州市中心医院(温州医科大学 定理临床学院)泛血管疾病管理实验室,浙江 温州 325000温州市中心医院(温州医科大学 定理临床学院)泛血管疾病管理实验室,浙江 温州 325000||温州市中心医院(温州医科大学 定理临床学院)内分泌科,浙江 温州 325000温州市中心医院(温州医科大学 定理临床学院)血管外科,浙江 温州 325000||温州市中心医院(温州医科大学 定理临床学院)泛血管疾病管理实验室,浙江 温州 325000温州市中心医院(温州医科大学 定理临床学院)血管外科,浙江 温州 325000||温州市中心医院(温州医科大学 定理临床学院)泛血管疾病管理实验室,浙江 温州 325000温州市中心医院(温州医科大学 定理临床学院)血管外科,浙江 温州 325000||温州市中心医院(温州医科大学 定理临床学院)泛血管疾病管理实验室,浙江 温州 325000温州市中心医院(温州医科大学 定理临床学院)血管外科,浙江 温州 325000||温州市中心医院(温州医科大学 定理临床学院)泛血管疾病管理实验室,浙江 温州 325000
生物科学
内皮祖细胞血管生成素微小RNA慢性下肢缺血
endothelial progenitor cellangiopoietinmicroRNAchronic lower limb ischemia
《基础医学与临床》 2026 (1)
46-55,10
浙江省卫生健康委医药卫生科技计划(2020KY295)
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