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

摘要/Abstract

摘要：

目的探讨过表达环状RNA（circRNAs）修饰牙髓干细胞（DPSCs）来源外泌体（Exo）促进人脐静脉内皮细胞（HUVECs）血管生成在牙髓血管再生中的作用，并阐明相关分子机制。方法分离乳齿源-、成人智齿源-和老年人恒牙源-原代DPSCs。流式细胞术检测3种原代DPSCs表面标志物蛋白阳性表达情况。使用3种来源的DPSCs与HUVECs建立共培养体系，将细胞分为乳牙源-原代DPSCs组、成人智齿源-原代DPSCs组和老年人恒牙源-原代DPSCs组。另将细胞分为Exo-OE vector组、Exo-circ_0026827 OE组和Exo-circRNA 124534 OE组，分别转染腺病毒介导的OE vector、circ_0026827 OE和circRNA124534 OE后，分离其细胞培养上清液Exo。细胞计数试剂盒8（CCK-8）法检测各组HUVECs增殖活性，实时荧光定量PCR（RT-qPCR）法检测各组Exo中血管生成相关circRNAs表达水平，Western blotting法检测共培养细胞上清中Exo标志物相关蛋白表达水平。验证各组细胞Exo摄取情况，血管生成-诱导实验检测细胞上清液中Exo分离情况。结果大部分细胞表面标志物表达情况为白细胞分化抗原44（CD44）（+）、白细胞分化抗原34（CD34）（-）和基质细胞抗原1（Stro-1）（+），鉴定为原代DPSCs。CCK-8法检测，与乳牙源-原代DPSCs组比较，成人智齿源-原代DPSCs组和老年人恒牙源-原代DPSCs组共培养的HUVECs增殖活性明显降低（P<0.01）；与成人智齿源-原代DPSCs组比较，老年人恒牙源-原代DPSCs组共培养的HUVECs增殖活性明显降低（P<0.01）。3种共培养体系细胞培养上清液Exo中白细胞分化抗原9（CD9）、热激蛋白70（HSP70）和肿瘤易感基因101（TSG101）均表达阳性。3种共培养体系细胞上清液中Exo的粒径为50~110 nm。RT-qPCR法检测，与乳牙源-原代DPSCs组比较，成人智齿源-原代DPSCs组和老年人恒牙源-原代DPSCs组Exo中circRNA124534和circ_0026827 mRNA表达水平均明显降低（P<0.01）；与成人智齿源-原代DPSCs组比较，老年人恒牙源-原代DPSCs组Exo中circRNA124534和circ_0026827 mRNA表达水平均明显降低（P<0.01）；3组Exo中circ_信号诱导增殖相关基因1（SIPA1L1） mRNA表达水平比较差异无统计学意义（P>0.05）。Western blotting法检测，与Exo-OE vector组比较，Exo-circ_0026827 OE组HUVECs中磷酸化p38丝裂原活化蛋白激酶（p-p38 MAPK）、血管内皮生长因子A（VEGF-A）、血管内皮生长因子受体2（VEGFR2）、血管发生素1（Ang-1）、基质细胞衍生因子1（SDF-1）和基质金属蛋白酶9（MMP-9）蛋白表达水平均明显升高（P<0.01）；与Exo-OE vector组比较，Exo-circRNA124534 OE组HUVECs细胞中p-p38 MAPK、VEGF-A、VEGFR2、Ang-1、SDF-1和MMP-9蛋白表达水平均明显升高（P<0.01）。各组HUVECs均成功摄取Exo。Exo-OE vector组HUVECs呈现较为平铺的生长状态；与Exo-OE vector组比较，Exo-circ_0026827 OE组和Exo-circRNA124534 OE组HUVECs呈现网状排列的生长状态，并有排列成管状的趋势。结论过表达circ_0026827和circRNA124534修饰乳牙DPSCs来源的Exo对HUVECs血管生成有一定促进作用。

关键词: 牙髓血管再生, 牙髓干细胞, 人脐静脉内皮细胞, 环状RNAs, 外泌体

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

中图分类号:

R781.3

刘景,王艳,黄旭. 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[J]. 吉林大学学报(医学版), 2025, 51(6): 1561-1570.

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.

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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