生信分析筛选间充质干细胞外泌体中与血管新生相关微小RNAs及黄芪甲苷干预的研究

Screening of neovascularization-related microRNAs in exosomes of mesenchymal stem cells by bioinformatics analysis and the intervention of astragaloside Ⅳ

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。

Objective To explore the effect of astragaloside Ⅳ(AS-Ⅳ) on the expression of neovascularization-related miRNAs in the exosomes(Exos) of human umbilical cord blood mesenchymal stem cells(hUCBMSCs) screened by bioinformatics analysis. Methods The miRNAs highly related to neovascularization in hUCBMSCs-Exos was screened by bioinformatics analysis. The hUCBMSCs were randomly divided into experimental and control groups. The experimental group was given DMEM/F12 medium containing 300 mg·L^(-1)AS-Ⅳ for 24 hours, and the control group was added with the same amount of phosphate buffer solution. The concentration of Exos was detected by dioctyl butyric acid method, and the expression of miRNAs was detected by reverse transcription-polymerase chain reaction. Results The concentrations of Exos in the experimental and control groups were(1.22±0.02) and(0.88±0.03) μg·μL^(-1), the difference was statistically significant(P<0.01). The expression levels of miRNA-126-3 p,miRNA-21,miRNA-214-5 p,miRNA-126-5 p,miRNA-145,miRNA-16-5 p and miRNA-195-5 p in the experimental group were 6. 69 ± 0. 57,1. 06 ± 0. 16,1. 06 ± 0. 21,0. 68 ± 0. 05,0. 51 ± 0. 17,0. 48 ± 0. 14 and 0. 46 ± 0. 10,the expression levels in the control group were 1. 04 ± 0. 14,0. 74 ± 0. 10,0. 48 ± 0. 14,1. 04 ± 0. 14,1. 02 ± 0. 17,1. 09 ± 0. 15 and 1. 18 ± 0. 20,the differences were statistically significant( all P <0. 05). Conclusion AS-Ⅳ can improve the ability of h UCBMSCs to secrete Exos that loaded with neovascularization-related miRNAs,showing a strong potential to induce neovascularization.