[1]边晓玮,张翠萍,李炳旻,等.胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响[J].第三军医大学学报,2020,42(12):1163-1173.
 BIAN Xiaowei,ZHANG Cuiping,LI Bingmin,et al.Effects of exosomes derived from placental mesenchymal stem cells on high glucose-induced senescence in human dermal fibroblasts [J].J Third Mil Med Univ,2020,42(12):1163-1173.
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胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第12期
页码:
1163-1173
栏目:
基础医学
出版日期:
2020-06-30

文章信息/Info

Title:
Effects of exosomes derived from placental mesenchymal stem cells on high glucose-induced senescence in human dermal fibroblasts
 
作者:
边晓玮张翠萍李炳旻马奎杨劼付小兵
天津医科大学研究生院;中国人民解放军总医院创新医学研究部组织修复与再生研究中心,皮肤损伤修复与再生北京市重点实验室
 
Author(s):
BIAN Xiaowei ZHANG Cuiping LI Bingmin MA Kui YANG Jie FU Xiaobing

Graduate School, Tianjin Medical University, Tianjin, 300070; 2Research Center for Tissue Repair and Regeneration,  Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, Division of innovative Medical Research, Chinese PLA General Hospital, Beijing, 100048, China

关键词:
外泌体胎盘间充质干细胞成纤维细胞高糖细胞衰老细胞增殖细胞迁移
Keywords:
exosomes placental mesenchymal stem cells fibroblasts high glucose cell senescence cell proliferation cell migration
分类号:
R322.99;R329.28;R587.102
文献标志码:
A
摘要:

目的探索高糖(high glucose, HG)促进体外培养的人皮肤成纤维细胞(human dermal fibroblasts, HDFs)的衰老条件,建立高糖老化模型;观察胎盘间充质干细胞来源的外泌体(exosomes, Exos)对高糖培养的成纤维细胞增殖、迁移及衰老的影响。方法分离人包皮组织的HDFs。实验分为3组:正常对照组(5.5 mmol/L葡萄糖)、HG1组(26 mmol/L葡萄糖)、HG2组(35 mmol/L葡萄糖)。CCK-8试剂盒检测各组细胞在1、3、5、7 d的增殖情况;各组培养7 d后进行划痕实验,观察各组HDFs在24 h内的迁移率;2′,7′二氯荧光素二醋酸(DCFH)法检测各组培养7 d后细胞内活性氧簇(ROS)水平;细胞衰老β-半乳糖苷酶染色试剂盒检测各组培养7 d后β-半乳糖苷酶活性。体外培养胎盘间充质干细胞(placenta mesenchymal stem cells, PMSCs),差速高速离心法分离Exos。将上述培养的HDFs分为3组:HG2组(35 mmol/L葡萄糖)、HG2+低浓度Exos(5 μg/mL)组、HG2+高浓度Exos(50 μg/mL)组,培养3 d后,检测细胞增殖情况、迁移率、ROS水平和β-半乳糖苷酶活性;流式细胞术检测细胞周期;Western blot分析衰老相关蛋白p53和p21表达情况。 结果与对照组相比,HG1和HG2组培养的HDFs增殖、迁移能力明显下降(P<0.01),ROS水平显著升高(P<0.01),β-半乳糖苷酶活性显著增加(P<0.01),而且各指标尤其以HG2组变化最为明显;Exos实验结果显示:高浓度Exos明显促进高糖培养HDFs的增殖和迁移(P<0.01),显著降低细胞内的ROS水平和β-半乳糖苷酶活性(P<0.01),同时使细胞内p53和p21蛋白的表达水平显著下降(P<0.01),而低浓度Exos无明显效果。结论胎盘间充质干细胞来源的Exos促进高糖培养的HDFs增殖、迁移,缓解细胞的氧化应激损伤和衰老状态,其机制可能与衰老相关蛋白p53和p21表达下调有关。
 

Abstract:

ObjectiveTo explore the optimal conditions for inducing senescence in human dermal fibroblasts (HDFs) cultured in high glucose and observe the effect of exosomes derived from human placental mesenchymal stem cells (PMSCs) on the proliferation, migration and senescence of high glucose-induced fibroblasts. MethodsHDFs were isolated from human foreskin tissue and cultured in the presence of 5.5 mmol/L glucose (normal control group), 26 mmol/L glucose (HG1 group) and 35 mmol/L glucose (HG2 group). CCK-8 assay was used to detect cell proliferation in each group on days 1, 3, 5 and 7 of cell culture. The cells cultured for 7 d were tested for migration within 24 h using cell scratch assay; reactive oxygen species (ROS) production in the cells was detected using 2′,7′-dichlorofluorescein diacetic acid (DCFH) method, and β-galactosidase activity was analyzed using a β-galactosidase staining kit. The HDFs cultured in 35 mmol/L glucose for 7 d were exposed for 3 days to 5 μg/mL and 50 μg/mL exosomes, which were isolated from human PMSCs by differential ultracentrifugation, and the cell proliferation, migration, ROS levels and β-galactoside activity were assessed. Flow cytometry was used to analyze the cell cycle changes and Western blotting was performed to detect the expression of aging-related proteins p53 and p21 in the cells following treatment with the exosomes. ResultsCompared with the control cells, the HDFs in HG1 and HG2 groups exhibited significantly decreased proliferation and migration ability (P<0.01) and increased ROS production (P<0.01) and β-galactoside activity (P<0.01). Treatment with the exosomes obviously promoted the proliferation and migration of HDFs cultured in high glucose (P<0.01). At the concentration of 50 μg/mL, PMSC-derived exosomes significantly decreased ROS production and β-galactoside activity (P<0.01) and obviously down-regulated the expression levels of p53 and p21 (P<0.01) in the HDFs cultured in high glucose, whereas a low concentration (5 μg/mL) of the exosomes did not produce such effects. ConclusionExosomes derived from PMSCs promote the proliferation and migration and alleviate oxidative stress-induced damage and cell senescence in high glucose-induced HDFs, the mechanism of which may involve the down-regulation of the aging-related proteins p53 and p21.

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更新日期/Last Update: 2020-06-19