过表达circRNAs修饰牙髓干细胞来源的外泌体对人脐静脉内皮细胞血管生成的促进作用

doi:10.13481/j.1671-587X.20250612

Abstract

Abstract:

Objective To discuss the role of circular RNAs （circRNAs）-overexpressing modified dental pulp stem cells （DPSCs）-derived exosomes （Exo） in promoting angiogenesis of the human umbilical vein endothelial cells （HUVECs） in dental pulp vascular regeneration， and to clarify the related molecular mechanism. Methods The primary DPSCs derived from deciduous teeth， adult wisdom teeth， and elderly permanent teeth were isolated. Flow cytometry was used to detect the positive expression of surface marker proteins in three kinds of primary DPSCs. Co-culture systems were established using three sources of DPSCs and HUVECs， and the cells were divided into deciduous teeth-derived primary DPSCs group， adult wisdom teeth-derived primary DPSCs group， and elderly permanent teeth-derived primary DPSCs group. Additionally， the cells were divided into Exo-OE vector group， Exo-circ_0026827 OE group， and Exo-circRNA124534 OE group； after transfection with adenovirus-mediated OE vector， circ_0026827 OE， and circRNA124534 OE， respectively， the Exo from the cell culture supernatant were isolated. Cell counting kit-8 （CCK-8） method was used to detect the proliferation activities of the HUVECs in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of angiogenesis-related circRNAs in the Exo in various groups； Western blotting method was used to detect the expression levels of Exo marker-related proteins in the supernatant of co-cultured cells. The uptakes of Exo by the cells in various groups were verified； angiogenesis induction experiment was used to detect the isolation of Exo from cell supernatant. Results Most cells expressed CD44（+）， CD34（-）， and Stro-1（+）， and were identified as primary DPSCs.The CCK-8 assay results showed that compared with deciduous teeth-derived primary DPSCs group， the proliferation activity of the HUVECs co-cultured in adult wisdom teeth-derived primary DPSCs group and elderly permanent teeth-derived primary DPSCs group was significantly decreased （P<0.01）； compared with adult wisdom teeth-derived primary DPSCs group， the proliferation activity of the HUVECs co-cultured in elderly permanent teeth-derived primary DPSCs group was significantly decreased （P<0.01）. cluster of differentiation 9 （CD9）， heat shock protein 70 （HSP70）， and tumor susceptibility gene 101（TSG101） were positively expressed in the Exo from the cell culture supernatant of three co-culture systems. The particle sizes of Exo from the cell supernatant in three co-culture systems were 50-110 nm. The RT-qPCR results showed that compared with deciduous teeth-derived primary DPSCs group， the mRNA expression levels of circRNA124534 and circ_0026827 in the Exo in adult wisdom teeth-derived primary DPSCs group and elderly permanent teeth-derived primary DPSCs group were significantly decreased （P<0.01）； compared with adult wisdom teeth-derived primary DPSCs group， the mRNA expression levels of circRNA124534 and circ_0026827 in the Exo in elderly permanent teeth-derived primary DPSCs group were significantly decreased （P<0.01）； there was no statistically significant difference in the circ_signal-induced proliferation-associated 1 like 1 （SIPA1L1） mRNA expression level among three groups （P>0.05）. The Western blotting results showed that compared with Exo-OE vector group， the protein expression levels of phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK）， vascular endothelial growth factor A （VEGF-A）， vascular endothelial growth factor receptor 2 （VEGFR2）， angiopoietin 1 （Ang-1）， stromal cell-derived factor 1 （SDF-1）， and matrix metalloproteinase 9 （MMP-9） in the HUVECs in overexpression circ_0026827 and circRNA124534 were significantly increased （P<0.01）； compared with Exo-OE vector group， the protein expression levels of p-p38 MAPK， VEGF-A， VEGFR2， Ang-1， SDF-1， and MMP-9 in the HUVECs in Exo-circRNA124534 OE group were significantly increased （P<0.01）. The HUVECs in various groups successfully took up Exo.The HUVECs in Exo-OE vector group showed a relatively flattened growth state. Compared with Exo-OE vector group， the HUVECs in Exo-circ_0026827 OE group and Exo-circRNA124534 OE group showed a reticularly arranged growth state and a tendency to form tubular structures. Conclusion The deciduous teeth DPSCs-derived Exo modified by overexpression circ_0026827 and circRNA124534 has a certain promoting effect on angiogenesis of the HUVECs.

Key words: Dental pulp vascular regeneration, Dental pulp stem cells, Human umbilical vein endothelial cells, Circular RNAs, Exosomes

CLC Number:

R781.3

Jing LIU,Yan WANG,Xu HUANG. Promoting effect of overexpressed circRNAs-modified dental pulp stem cell-derived exosomes on angiogenesis of human umbilical vein endothelial cells[J].Journal of Jilin University(Medicine Edition), 2025, 51(6): 1561-1570.

Figures/Tables 8

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References 29

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Group	CircRNA124534	Circ_SIPA1L1	Circ_0026827
Deciduous teeth derived-primary DPSCs	1.00±0.22	1.00±0.17	1.00±0.18
Adult wisdom teeth derived-primary DPSCs	0.44±0.08^*	1.17±0.24	0.57±0.05^*
Elderly permanent teeth derived-primary DPSCs	0.15±0.04^*△	1.11±0.13	0.26±0.03^*△
F	59.590	1.294	69.440
P	<0.01	0.303	<0.01

Group	p-p38 MAPK	VEGF-A	VEGFR2	Ang-1	SDF-1	MMP-9
Exo-OE vector	1.00±0.13	1.00±0.11	1.00±0.09	1.00±0.10	1.00±0.11	1.00±0.14
Exo-circ_0026827 OE	2.11±0.19	1.64±0.16	1.82±0.15	1.57±0.12	1.33±0.08	3.24±0.27
t	11.81	8.07	11.48	8.94	5.94	18.04
P	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

Group	p-p38 MAPK	VEGF-A	VEGFR2	Ang-1	SDF-1	MMP-9
Exo-OE vector	1.00±0.11	1.00±0.16	1.00±0.10	1.00±0.14	1.00±0.13	1.00±0.15
Exo-circRNA124534 OE	2.84±0.25	1.81±0.17	1.37±0.14	1.78±0.12	1.87±0.18	2.26±0.21
t	16.50	8.50	5.27	10.36	9.60	11.96
P	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

Promoting effect of overexpressed circRNAs-modified dental pulp stem cell-derived exosomes on angiogenesis of human umbilical vein endothelial cells

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