炎症微环境中脐带干细胞外泌体对牙周膜干细胞增殖和迁移的影响

田萧羽; 杨烁; 朱彪; 赵立升; 李琳; 廖思达; 温宁

doi:10.3969/j.issn.2095-5227.2021.05.012

炎症微环境中脐带干细胞外泌体对牙周膜干细胞增殖和迁移的影响

Exosomes derived from umbilical cord mesenchymal stem cells promote proliferation and migration of periodontal ligament stem cells in inflammatory microenvironment

摘要

摘要:
背景在炎症微环境中牙周膜干细胞(periodontal ligament stem cells，PDLSCs)的生物活性受到抑制是牙周再生困难的重要原因。脐带间充质干细胞(umbilical cord mesenchymal stem cells，UCMSCs)来源外泌体(exosome，Exo)具有与UCMSCs相似的生物学作用，广泛用于多种组织的再生修复，但在牙周再生方面的研究仍然缺乏，对炎症微环境中PDLSCs生物活性的影响尚不明确。
目的探讨在炎症微环境中UCMSCs来源外泌体对PDLSCs增殖和迁移的影响。
方法提取UCMSCs来源外泌体并进行鉴定；分离PDLSCs并进行鉴定；荧光显微镜下观察PDLSCs对外泌体的摄取内吞；按照空白对照组、肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)组、TNF-α + 25 μg/mL外泌体组、TNF-α + 50 μg/mL外泌体组、TNF-α + 100 μg/mL外泌体组进行实验分组，分别通过CCK-8实验、划痕实验、Transwell实验检测炎症微环境中外泌体对PDLSCs增殖、横向迁移及纵向迁移的影响。测炎症微环境中外泌体对PDLSCs增殖、横向迁移及纵向迁移的影响。
结果透射电镜下UCMSCs来源外泌体呈现类圆型，直径100 nm左右。Western blot检测到外泌体表面蛋白CD9、CD63、CD81表达阳性。成功提取PDLSCs，能够表达间充质干细胞表面标记蛋白CD90、CD73、CD29、CD105。免疫荧光结果显示UCMSCs来源外泌体可以被PDLSCs内吞。CCK-8实验表明UCMSCs 来源外泌体可以促进炎症微环境中PDLSCs的增殖(P＜0.05)，100 μg/mL外泌体促增殖作用最佳(P＜0.05)。Transwell实验和划痕实验分别表明UCMSCs来源外泌体可以促进炎症微环境中PDLSCs的横向迁移及纵向迁移，且呈浓度依赖性，外泌体浓度100 μg/mL促迁移作用最佳(P＜0.05)。
结论在炎症微环境下，UCMSCs来源外泌体可以有效促进PDLSCs的增殖和迁移，高浓度外泌体促增殖及迁移效果更佳，这为外泌体应用于牙周再生奠定了基础。

Abstract:
Background Inhibition of the biological activity of periodontal ligament stem cells (PDLSCs) in an inflammatory microenvironment is an important cause for the difficulty of periodontal regeneration. The exosome (Exo) derived from umbilical cord stem cells (UCMSCs) has similar biological effects to UCMSCs and is widely used for the regeneration and repair of a variety of tissues, but research on periodontal regeneration is still lacking, and the effect on PDLSCs in the inflammatory microenvironment is not clear.
Objective To investigate the role of UCMSCs-derived exosomes in the proliferation and migration of PDLSCs under an inflammatory microenvironment.
Methods Exosomes were extracted and identified from UCMSCs. PDLSCs were isolated and identified. Uptake of exosomes by endocytosis of PDLSCs was observed under fluorescence microscope. The experimental groups were divided into blank control group, TNF-α group, TNF-α plus 25 μg/mL exosome group, TNF-α plus 50 μg/mL exosome group, and TNF-α plus 100 μg/mL exosome group. The effects of exosomes in inflammatory microenvironment on the proliferation, lateral and longitudinal migration of PDLSCs were detected by CCK-8 assay, scratch test and Transwell assay, respectively.
Results Under transmission electron microscopy, the morphology of exosomes from UCMSCs were round, with a diameter of about 100 nm. The expression of CD9, CD63 and CD81 on the surface of exosomes was positive detected by Western Blot. PDLSCs were successfully extracted and expressed MSCs-related surface markers including CD90, CD73, CD29, CD105. Immunofluorescence results showed that exosomes from UCMSCs could be endocytosed by PDLSCs. CCK-8 test results showed that UCMSCS-derived exosomes promoted the proliferation of PDLSCs under inflammatory microenvironment (P＜0.05), and the proliferation rate at the concentration of 100 μg/mL was the highest (P＜0.05). Transwell test and scratch test showed that the UCMSCS-derived exosomes promoted the migration of PDLSCs laterally and longitudinally in the inflammatory microenvironment in a concentration-dependent manner, with the highest rate at the concentration of 100 μg/mL (P＜0.05).
Conclusion Under the inflammatory microenvironment, UCMSCs-derived exosomes can effectively promote PDLSCs proliferation and migration, and high concentrations of exosomes promote proliferation and migration more effectively.

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