小肠黏膜下层处理的间充质干细胞片促进伤口愈合

Umbilical cord mesenchymal stem cells sheets treated by small intestinal submucosa promote wound healing

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。

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.