托里消毒散对内皮祖细胞外泌体中表达与炎症相关miRNAs的影响*

作者：黄鸿宇1，郭子莘1，陈甲尧1，胡清睿1，钟星星1，危 洋1，李勤霞1，黄子歆2，熊 武3，张 熙1

单位：1.湖南中医药大学中西医结合学院，湖南 长沙 410208； 2.湖南中医药大学中医学院，湖南 长沙 410208； 3.湖南中医药大学第一附属医院，湖南 长沙 410007

引用：引用：黄鸿宇，郭子莘，陈甲尧，胡清睿，钟星星，危洋，李勤霞，黄子歆，熊武，张熙.托里消毒散对内皮祖细胞外泌体中表达与炎症相关miRNAs的影响[J].中医药导报,2025,31(5):45-50.

DOI：10.13862/j.cn43-1446/r.2025.05.008

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摘要：

目的：探讨托里消毒散浸提液促进内皮祖细胞（EPCs）增殖的最佳质量浓度，进一步分析托里消毒散浸提液对EPCs分泌外泌体（EPC-Exos）及外泌体中与炎症相关微小RNA（miRNAs）表达的影响。方法：无菌条件下取足月健康新生儿脐带血，分离得到单个核细胞，进一步分离并鉴定得到EPCs，将鉴定成功的EPCs用不同质量浓度梯度（0.00、0.01、0.05、0.10、0.20、0.50、0.80、1.00、2.00、3.00、4.00、8.00、16.00、32.00、48.00、64.00、100.00及128.00 mg/mL）的托里消毒散浸提液干预24 h，筛选出促进EPCs增殖的最佳质量浓度。另将鉴定成功的EPCs分为托里消毒散浸提液组（实验组）和磷酸盐缓冲液（PBS）组（对照组），实验组用最佳质量浓度的托里消毒散浸提液干预，对照组用等体积的PBS液处理，培养24 h，分别收集两组细胞外泌体。Western blotting法检测两组外泌体的生物学标记物（CD81、CD63、CD9、Calnexin）；采用透射电子显微镜观察外泌体的形态；利用纳米粒子追踪分析技术检测外泌体的粒径；BCA检测两组外泌体质量浓度；qPCR法检测各组外泌体中与炎症相关miRNAs分子的表达。结果：托里消毒散浸提液促进EPCs增殖的最佳质量浓度为0.50 mg/mL；各组外泌体的生物学标记物（CD81、CD63、CD9）的表达均呈阳性，Calnexin的表达呈阴性。两组外泌体均形态完整、呈球形、大小均一，对照组中97.3%的EPC-Exos粒径为41.1~109.0 nm；实验组中99.5%的EPC-Exos粒径为120.3~284.5 nm。对照组与实验组EPC-Exos总蛋白质量浓度分别为（1.16±0.01） μg/μL和（1.04±0.02） μg/μL，差异均有统计学意义（P＜0.01）。实验组中部分与炎症相关miRNAs表达较对照组增多，即miR-92a-3p和miR-92a-5p表达增多，miR-92a-3p差异无统计学意义（P＞0.05），miR-92a-5p差异有统计学意义（P＜0.05），而miR-30d-3p、miR-30d-5p、miR-144-3p和miR-144-5p的表达较对照组明显减少，差异均有统计学意义（P＜0.05）。结论：托里消毒散浸提液在促进内皮祖细胞增殖的同时能促进其分泌外泌体，且所分泌的外泌体负载调控炎症反应的miRNAs。托里消毒散浸提液能介导EPC-Exos发挥炎症调控的作用。

关键词：托里消毒散；内皮祖细胞；外泌体；微小RNA；炎症

Abstract：

Objective: To investigate the optimal concentration of Tuoli Xiaodu powder extracts for endothelial progenitor cells (EPCs) proliferation activity, to further analyze the effect of Tuoli Xiaodu powder extracts on the secretion of EPCs exosomes (EPC-Exos) and the expression of inflammation-related Micro RNAs (miRNAs) in the exosomes. Methods: Term healthy newborn umbilical cord blood was extracted under aseptic conditions. Mononuclear cells were isolated, and EPCs were further isolated and identified. The successful EPCs were treated with different concentration gradients (0.00, 0.01, 0.05, 0.10, 0.20, 0.50, 0.80, 1.00, 2.00, 3.00, 4.00, 8.00, 16.00, 32.00, 48.00, 64.00, 100.00, 128.00 mg/mL) of Tuoli Xiaodu powder extracts was treated for 24 h, and the optimal concentration of promoting EPCs proliferation was screened. In addition, EPCs successfully identified were divided into Tuoli Xiaodu powder extracts group (experimental group) and phosphate buffer (PBS) group (control group). The experimental group was treated with the optimal concentration of Tuoli Xiaodu powder extracts, and the control group was treated with equal volume of PBS solution for 24 h. The exosomes of the two groups were collected respectively. The biological markers (CD81, CD63, CD9, Calnexin) of the two groups of exosomes were detected by Western blotting. The morphology of exosomes was observed by transmission electron microscope. The particle size of exosomes was detected by nanoparticle tracking analysis. The concentrations of exosomes in the two groups were detected by BCA. The expression of inflammation-related miRNAs in exosomes of each group was detected by qPCR. Results: The optimal concentration of Tuoli Xiaodu powder to promote EPCs proliferation was 0.50 mg/mL. The expression of biological markers of exosomes (CD81, CD63, CD9) was positive in two groups, while the expression of Calnexin was negative. The exosomes in two groups were intact, spherical and uniform in size, and 97.3% of EPC-Exos in the control group had a particle size of (41.1-109.0) nm. The particle size of 99.5% EPC-Exos in the experimental group was (120.3-284.5) nm. The total protein concentration of EPC-Exos was (1.16±0.01) μg/μL and (1.04±0.02) μg/μL in control group and experimental group, respectively, with significant differences (P<0.01). The expression of some inflammation-related miRNAs increased in experimental group compared with that in the control group. The expression of miR-92a-3p and miR-92a-5p was increased. There was no statistical significance for mir-92a-3p (P>0.05), while there was statistical significance for miR-92a-5p (P<0.05). However, the expressions of miR-30d-3p, miR-30d-5p, miR-144-3p and miR-144-5p were significantly decreased in experimental group, compared with the control group, with significant differences (P<0.05). Conclusion: Tuoli Xiaodu powder extracts can promote the proliferation of endothelial progenitor cells and promote their secretion of exosomes, and the secretory exosomes are loaded with miRNAs that regulate inflammatory response. It indicates that Tuoli Xiaodu powder extracts can mediate EPC-Exos to play a role in inflammation regulation.

Key words：Tuoli Xiaodu powder; endothelial progenitor cells; exosome; miRNAs; inflammation

发布时间：2025-12-30

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