<br>人脱落乳牙 (SHED) 干细胞及其细胞外囊泡的分泌组可改善角质形成细胞迁移、活力并减弱 H2O2 诱导的细胞毒性,Wound Repair and Regeneration

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人脱落乳牙 (SHED) 干细胞及其细胞外囊泡的分泌组可改善角质形成细胞迁移、活力并减弱 H2O2 诱导的细胞毒性
Wound Repair and Regeneration ( IF 3.8 ) Pub Date : 2023-12-01 , DOI: 10.1111/wrr.13131
Juliana Girón Bastidas _{1,

2} , Natasha Maurmann _{1,

2} , Juliete Nathali Scholl ₃ , Augusto Ferreira Weber ₃ , Raíssa Padilha Silveira ₁ , Fabricio Figueiró _{3,

4} , Marco Augusto Stimamiglio ₅ , Bruna Marcon ₅ , Alejandro Correa ₅ , Patricia Pranke _{1,

2,

6}

Affiliation

在临床前研究中，使用间充质干细胞/基质细胞的分泌组和细胞外囊泡（EV）进行伤口愈合的疗法已被证明是成功的。本研究旨在表征人脱落乳牙 (SHED) 干细胞分泌蛋白的含量，并分析 SHED 条件培养基 (SHED-CM) 和 SHED 细胞外囊泡 (SHED-EV) 对角质形成细胞的体外影响。对电动汽车进行了分离和表征。评估了用 SHED-EV 和条件培养基 (CM) 处理的细胞的角质形成细胞活力和迁移。使用H ₂ O ₂进行体外H ₂ O ₂诱导的HaCaT细胞凋亡模型，然后进行3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑(MTT)和活/死测定。最后，通过免疫荧光染色评估并通过 RT-qPCR 确认经分泌组和 EV 处理的角质形成细胞中血管内皮生长因子 (VEGF) 的表达。 SHED-EVs 显示出杯形形态，表达外泌体 CD9 和 CD63 的经典标记，直径为 181 ± 87 nm。通过荧光显微镜证实了 HaCaT 细胞对 EV 的内化。蛋白质组学分析发现，SHED-CM 富含与应激反应和发育相关的蛋白质，包括细胞因子（CXCL8、IL-6、CSF1、CCL2）和生长因子（IGF2、MYDGF、PDGF）。结果还表明，50% CM 和 0.4–0.6 μg/mL EV 对于改善角质形成细胞活力、迁移和减弱 H ₂ O ₂诱导的细胞毒性同样有效。此外，当用 SHED 分泌组和 EV 处理时，角质形成细胞上 VEGF 的表达增加。此外，当用 SHED 分泌组和 EV 处理时，角质形成细胞中的 VEGF 基因表达显着增加。因此，SHED-CM 和 SHED-EV 都可能成为加速伤口愈合中上皮再形成的有前途的治疗工具。

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Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H2O2-induced cytotoxicity

Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H₂O₂ in vitro was performed with H₂O₂ followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4–0.6 μg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H₂O₂-induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.

更新日期：2023-12-01

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