小肠黏膜下层处理的间充质干细胞片促进伤口愈合

doi:10.6040/j.issn.1671-7554.0.2024.0185

摘要/Abstract

摘要： 目的探讨细胞外基质(extracellular matrix, ECM),尤其是小肠黏膜下层(small intestinal submucosa, SIS)处理后的脐带间充质干细胞(umbilical cord mesenchymal stem cells, UC-MSCs)细胞片对伤口愈合的影响。方法使用组织块贴壁法从脐带组织中分离、培养原代UC-MSCs。化学脱核法制备脱细胞SIS,通过物理粘附方式将重组人纤维连接蛋白(recombinant human fibronectin, rFN)、SIS和基质胶(Matrigel)固定在聚苯乙烯(polystyrene, PS)细胞培养皿表面。分析PS表面、rFN-PS表面、SIS-PS表面以及Matrigel-PS表面对UC-MSCs形态的影响,RT-PCR法检测基因表达的变化,使用纳米颗粒追踪分析检测细胞外囊泡(extracellular vesicles, EVs)分泌情况的改变。分别用无ECM处理的、rFN处理的、SIS处理的以及Matrigel处理的UC-MSCs细胞片治疗大鼠皮肤全层切除创面,通过计算伤口面积占原始伤口面积的比值进一步验证治疗效果。结果与PS表面相比,SIS-PS表面培养的UC-MSCs形态未受影响(P>0.05);RT-PCR检测结果显示,SIS-PS表面培养的UC-MSCs中NANOG同源框(nanog homeobox, NANOG,P=0.041)、SRY-Box 转录因子-2(SRY-box transcription factor 2,SOX-2,P=0.009)、八聚体结合转录因子-4(octamer-binding transcription factor-4,OCT-4,P<0.001)、白介素-10(interleukin-10,IL-10,P=0.049)、吲哚胺2, 3-双加氧酶(indoleamine 2,3-dioxygenase,IDO,P=0.007)和转化生长因子-β(transforming growth factor beta,TGF-β,P=0.046)基因表达增强,而且分泌EVs的能力显著提高(P<0.001)。动物实验结果可见,使用SIS处理的UC-MSCs细胞片治疗组对伤口愈合的促进作用最为显著,第7天伤口占原始伤口面积的比值最低为26.9%±6.1%。结论 SIS-PS表面培养的UC-MSCs显著提高了免疫抑制和囊泡分泌能力,由此制成的细胞冻干片具有更强的促进伤口愈合的能力。

关键词: 间充质干细胞, 小肠黏膜下层, 伤口愈合, 细胞外囊泡, 胞外基质

Abstract: Objective To explore the effect of extracellular matrix(ECM), especially the small intestinal submucosa(SIS)-treated umbilical cord mesenchymal stem cells(UC-MSCs)sheets on wound healing. Methods Primary UC-MSCs were isolated and cultured from umbilical cord tissues using the tissue block apposition method. Decellularized SIS was obtained using a chemical method, and recombinant human fibronectin(rFN), SIS, and Matrigel were immobilized on the surface of polystyrene(PS)cell culture dishes by physical adhesion. The effects of PS surface, rFN-PS surface, SIS-PS surface, and Matrigel-PS surface on the morphology of UC-MSCs were investigated, and the changes in gene expression were detected by RT-PCR, then changes in the secretion of extracellular vesicles(EVs)were detected using nanoparticle tracking analysis. Rat skin total excision wounds were treated with ECM-free, rFN-treated, SIS-treated, and Matrigel-treated UC-MSCs cell sheets, respectively, and the therapeutic effect was further verified by calculating the ratio of wound area to the original wound area. Results Compared with the PS surface, the morphology of UC-MSCs cultured on the SIS-PS surface was almost unaltered(P>0.05), and RT-PCR assay showed that gene expression of the SIS-PS surface-cultured UC-MSCs was enhanced, such as Nanog homeobox(NANOG, P=0.041), SRY-box transcription factor 2(SOX-2, P=0.009), octamer-binding transcription factor-4(OCT-4, P<0.001), interleukin-10(IL-10, P=0.049), indoleamine 2,3-dioxygenase(IDO, P=0.007), and transforming growth factor beta(TGF-β, P=0.046), meanwhile the ability of the SIS-PS surface-cultured UC-MSCs to secrete EVs was significantly increased(P<0.001). The results of the animal experiments revealed that the lowest percentage of wound to original wound area was 26.9%±6.1% in the group of animals treated with SIS-treated UC-MSCs cell sheets at day 7. Conclusion UC-MSCs cultured on the SIS-PS surface significantly improved immunosuppressive function and EVs secretion ability, and the corresponding cell sheets have a stronger ability to promote wound healing.

Key words: Mesenchymal stem cells, Small intestinal submucosa, Wound healing, Extracellular vesicles, Extracellular matrix

中图分类号:

R318.08

沈飞飞,李栋,刘玲红,鞠秀丽. 小肠黏膜下层处理的间充质干细胞片促进伤口愈合[J]. 山东大学学报 (医学版), 2024, 62(4): 68-77.

SHEN Feifei, LI Dong, LIU Linghong, JU Xiuli. Umbilical cord mesenchymal stem cells sheets treated by small intestinal submucosa promote wound healing[J]. Journal of Shandong University (Health Sciences), 2024, 62(4): 68-77.

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