hUCMSC-sEVs对hDPSCs成骨/成牙分化及HUVECs成管能力的影响

doi:10.19439/j.sjos.2025.06.001

摘要/Abstract

摘要： 目的:探讨人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）来源的小细胞外囊泡（small extracellular vesicles,sEVs）对人牙髓干细胞（human dental pulp stem cells,hDPSCs）迁移和成骨/成牙分化能力、人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）迁移和成管能力的影响及其潜在机制。方法:培养及鉴定hUCMSCs、hDPSCs和HUVECs,提取并鉴定hUCMSC-sEVs及LPSpre-hUCMSC-sEVs。按阴性对照组（NC）、阳性对照组（PC）、hUCMSC-sEVs组及LPSpre-hUCMSC-sEVs组处理HUVECs及hDPSCs,利用Transwell及划痕实验检测细胞迁移能力,管形成实验检测HUVECs的成管能力;茜红素染色、RT-qPCR检测hDPSCs成骨/成牙分化能力;小RNA高通量测序检测sEVs的miRNA表达谱。结果:成功分离并鉴定hUCMSCs、hDPSCs和HUVECs,提取并鉴定hUCMSC-sEVs和LPSpre-hUCMSC-sEVs。与NC组相比,LPSpre-hUCMSC-sEVs组及hUCMSC-sEVs组均可增强hDPSCs、HUVECs迁移及HUVECs小管形成能力（P<0.05）。在促hDPSCs迁移中,LPSpre-hUCMSC-sEVs组及hUCMSC-sEVs组差异无统计学意义（P>0.05）,而其他作用LPSpre-hUCMSC-sEVs组强于hUCMSC-sEVs组（P<0.05）。LPSpre-hUCMSC-sEVs组及hUCMSC-sEVs组钙盐沉积量高于阳性对照组,且LPSpre-hUCMSC-sEVs组钙盐沉积量高于hUCMSC-sEVs组（P<0.05）。PC组、hUCMSC-sEVs组及LPSpre-hUCMSC-sEVs组的ALP、OSX、OCN及RUNX2的mRNA表达水平高于NC组（P<0.05）,且hUCMSC-sEVs组与LPSpre-hUCMSC-sEVs组高于PC组（P<0.05）。LPSpre-hUCMSC-sEVs组OCN 及RUNX2 表达水平高于hUCMSC-sEVs组（P<0.05）,但ALP、OSX差异无统计学意义（P>0.05）。PC组及hUCMSC-sEVs组的DSPP表达高于NC组,但差异无统计学意义（P>0.05）。LPSpre-hUCMSC-sEVs组DSPP表达水平高于PC组（P<0.05）。sEVs测序检测出共同高表达的hsa-miR-21-5p、hsa-let-7a-5p、hsa-miR-100-5p、hsa-miR-26a-5p、hsa-miR-222-3p及差异表达的hsa-miR-199a-3p、hsa-miR-122-5p、hsa-miR-1246、hsa-miR-615-3p等可能是LPSpre-hUCMSC-sEVs发挥作用的关键因子。结论:hUCMSC-sEVs促进hDPSCs迁移及成骨/成牙分化、HUVECs迁移及血管生成,脂多糖预处理可促进上述作用,其机制可能与LPSpre-hUCMSC-sEVs中表达的miRNAs有关。

关键词: 脂多糖, 小细胞外囊泡, 人脐带间充质干细胞, 人牙髓干细胞, 人脐静脉内皮细胞, 成骨/成牙分化

Abstract: PURPOSE: To investigate the effects of small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stem cells(hUCMSCs) on the migration and osteogenic/odontogenic differentiation ability of human dental pulp stem cells(hDPSCs), the migration and tube formation ability of human umbilical vein endothelial cells (HUVECs) and their possible mechanisms. METHODS: hUCMSCs, hDPSCs and HUVECs were cultured and identified, hUCMSC-sEVs and LPSpre-hUCMSC-sEVs were isolated and identified, HUVECs and hDPSCs were assigned to 4 kinds of treatments, including the negative control group(NC), the positive control group(PC), the hUCMSC-sEVs group and the LPSpre-hUCMSC-sEVs group. Cell migration ability was detected by Transwell and wound healing assays. Tube formation capacity of HUVECs was assessed by tube formation experiment. The osteogenic/odontogenic differentiation ability of hDPSCs was evaluated by alizarin red staining and RT-qPCR. High-throughput small RNA sequencing was used to define miRNA profiles in sEVs. RESULTS: hUCMSCs, hDPSCs, HUVECs, hUCMSC-sEVs and LPSpre-hUCMSC-sEVs were successfully isolated and identified. Compared with NC group, both LPSpre-hUCMSC-sEVs group and hUCMSC-sEVs group promoted migration of hDPSCs, migration and tube formation of HUVECs. There was no significant difference between LPSpre-hUCMSC-sEVs group and hUCMSC-sEVs group in promoting migration of hDPSCs (P>0.05). LPSpre-hUCMSC-sEVs group was stronger than hUCMSC-sEVs group in promoting migration and tube formation of HUVECs(P<0.05). The calcium salt deposition in LPSpre-hUCMSC-sEVs group and hUCMSC-sEVs group was higher than that in PC group, and the calcium salt deposition in LPSpre-hUCMSC-sEVs group was higher than that in hUCMSC-sEVs group(P<0.05). The mRNA expression levels of ALP, OSX, OCN and RUNX2 in PC group, hUCMSC-sEVs group and LPSpre-hUCMSC-sEVs group were higher than those in NC group(P<0.05), and hUCMSC-sEVs group and LPSpre-hUCMSC-sEVs group were higher than PC group(P<0.05). In addition, the expression levels of OCN and RUNX2 in LPSpre-hUCMSC-sEVs group were higher than those in hUCMSC-sEVs group(P<0.05), while there was no significant difference in ALP and OSX between LPSpre-hUCMSC-sEVs group and hUCMSC-sEVs group(P>0.05). The expression level of DSPP in PC group and hUCMSC-sEVs group was higher than that in NC group, but the difference was not statistically significant(P>0.05). The expression level of DSPP in LPSpre-hUCMSC-sEVs group was higher than that in PC group (P<0.05). The most highly expressed miRNAs including hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-100-5p, hsa-miR-26a-5p and hsa-miR-222-3p, and differentially expressed miRNAs including hsa-miR-199a-3p, hsa-miR-122-5p, hsa-miR-1246 and hsa-miR-615-3p were detected, which may be the key factors of LPSpre-hUCMSC-sEVs. CONCLUSIONS: Small extracellular vesicles derived from human umbilical cord mesenchymal stem cells can promote migration and osteogenic/odontogenic differentiation of hDPSCs, as well as migration and angiogenesis of HUVECs, and LPS can enhance these effects, which may be related to miRNAs which are the most abundantly and diffferentially expressed in LPSpre-hUCMSC-sEVs.

Key words: Lipopolysaccharide, Small extracellular vesicles, Human umbilical cord mesenchymal stem cells, Human dental pulp stem cells, Human umbilical vein endothelial cells, Osteogenic/odontogenic differentiation

中图分类号:

R781.05

康京艺, 韦日霞, 邓惠丹, 李泉洁, 吴煜. hUCMSC-sEVs对hDPSCs成骨/成牙分化及HUVECs成管能力的影响[J]. 上海口腔医学, 2025, 34(6): 561-570.

Kang Jingyi, Wei Rixia, Deng Huidan, Li Quanjie, Wu Yu. The effects of hUCMSC-sEVs on osteogenic/odontogenic differentiation of hDPSCs and tube formation ability of HUVECs[J]. Shanghai Journal of Stomatology, 2025, 34(6): 561-570.

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