脐静脉内皮细胞外泌体和内皮祖细胞外泌体对hDPSCs增殖及迁移能力的比较研究

doi:10.13591/j.cnki.kqyx.2022.11.004

摘要/Abstract

摘要： 目的将人脐静脉内皮细胞外泌体(human umbilical vein endothelial cells-derived exosome,HUVECs-exo)和内皮祖细胞外泌体(endothelial progenitor cells-derived exosome,EPCs-exo)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)增殖和迁移能力的影响进行比较研究,以期为外泌体在牙髓再生中的应用积累经验。方法采用超速离心法分离提取外泌体,透射电镜观察外泌体形态,纳米粒子跟踪分析技术(nanoparticle tracking analysis,NTA)检测外泌体粒径大小,Western blot蛋白印迹检测外泌体标志蛋白CD9、CD63、TSG101的表达。将两种外泌体按照5、10、20 μg/mL浓度梯度分别作用于hDPSCs,通过CCK-8实验检测hDPSCs增殖能力,细胞划痕实验和Transwell实验检测hDPSCs迁移能力。结果透射电镜下观察两种外泌体均呈圆盘状,粒径集中在30～150 nm,阳性表达CD9、CD63、TSG101三种标志蛋白。与对照组(Control组)相比,两种外泌体在不同浓度下均对hDPSCs增殖能力无显著促进作用,差异无统计学意义(P>0.05)。细胞划痕实验和Transwell实验表明,与对照组相比,两种外泌体均可促进hDPSCs迁移,差异有统计学意义(P<0.05),其中10 μg/mL外泌体浓度作用效果最明显(P<0.01)。相同浓度的两种外泌体组间作用效果差异无统计学意义(P>0.05)。结论本实验成功分离提取到两种细胞来源的外泌体并进行鉴定,将不同浓度的两种细胞来源的外泌体作用于hDPSCs,发现对其增殖能力无明显促进作用,但可以促进其迁移,尤以10 μg/mL外泌体浓度促进迁移效果最为明显。

关键词: 脐静脉内皮细胞, 内皮祖细胞, 人牙髓干细胞, 外泌体, 牙髓再生

Abstract: Objective Human umbilical vein endothelial cells-derived exosome (HUVECs-exo) and endothelial progenitor cells-derived exosome (EPCs-exo) were co-cultured with hDPSCs respectively, to study and compare their effects on proliferation and migration of hDPSCs, in order to provide reference for the application of exosomes in pulp regeneration. Methods Exosomes derived from human umbilical vein endothelial cells and endothelial progenitor cells were isolated by ultracentrifugation in vitro. The morphologies of both exosomes were observed by transmission electron microscopy. Meanwhile, the size of exosomes was measured by NTA. Western blot was used to analyze the exosome surface markers of exosomes including CD9, CD63 and TSG101. Both exosomes were applied to human dental pulp stem cells according to the concentration gradient of 5, 10 and 20 μg/mL, respectively. The proliferation of dental pulp stem cells was detected by CCK-8 assay and migration ability of dental pulp stem cells was detected by cell scratch wound healing assay and Transwell assay. Results Under transmission electron microscope, both exosomes exhibited a discoid morphology, with particle size ranging from 30 nm to 150 nm. Western blot indicated the expression of exosome surface markers including CD9, CD63 and TSG101 in both exosomes. Compared with the control group, two kinds of exosomes with different concentrations didn't promote the proliferation of human dental pulp stem cells (P>0.05). The cell scratch wound healing test and Transwell test showed that both exosomes could promote the migration of human dental pulp stem cells compared with the control group, and the difference was statistically significant (P<0.05), in which the concentration of 10 μg/mL exosomes had the most significant effect (P<0.01). There was no significant difference in the effect of two exosomes (P>0.05). Conclusion Exosomes from the two kinds of cells did not significantly promote the proliferation of human dental pulp stem cells, but could improve the migration of human dental pulp stem cells, especially at the concentration of 10 μg/mL.

Key words: umbilical vein endothelial cells, endothelial progenitor cells, human dental pulp stem cells, exosome, dental pulp regeneration

中图分类号:

R349.5

张雪, 徐兆莹, 蒋鹏飞, 潘爽. 脐静脉内皮细胞外泌体和内皮祖细胞外泌体对hDPSCs增殖及迁移能力的比较研究[J]. 口腔医学, 2022, 42(11): 979-983.

ZHANG Xue, XU Zhaoying, JIANG Pengfei, PAN Shuang. Comparative study on the proliferation and migration of umbilical vein endothelial cells-derived exosome and endothelial progenitor cells-derived exosome on human dental pulp stem cells[J]. Stomatology, 2022, 42(11): 979-983.

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