年轻化间充质干细胞条件培养基促进小鼠创面愈合的研究

Rejuvenated mesenchymal stem cell conditioned medium in promoting wound healing in mice

背景间充质干细胞(mesenchymal stem cell,MSC)条件培养基无细胞疗法是创伤修复领域的一种新型治疗方案,但其治疗效果受间充质干细胞状态的影响。目的探究年轻化MSC培养基对小鼠创面愈合的作用。方法建立间充质干细胞复制性衰老(传代至15~20代)模型,检测其衰老指征;使用小分子化合物[丙戊酸(valproic acid,VPA)和RepSox]逆转衰老间充质干细胞并验证逆衰老效果。分别收集衰老MSC培养基与年轻化MSC培养基。用收集到的两种条件培养基培养人成纤维细胞,使用CCK-8实验、Ki-67免疫荧光、划痕实验和Transwell实验检测这两种条件培养基对成纤维细胞增殖、迁移和侵袭能力的影响。分别用衰老MSC培养基、年轻化MSC培养基和0.9%氯化钠注射液治疗C57BL/6小鼠背部皮肤全层缺损模型,评估3种治疗方法对创面愈合的影响。结果与衰老的间充质干细胞相比,经小分子处理后衰老间充质干细胞SA-β-gal阳性率降低(P<0.01),且免疫荧光检测显示其衰老基因P21、P16表达降低(P<0.01)。经年轻化MSC培养基处理的成纤维细胞增殖、迁移和侵袭能力增强(P<0.01)。创伤模型显示,与对照组相比,年轻化MSC培养基治疗组小鼠创面愈合速度加快(P<0.01);年轻化MSC培养基治疗组小鼠创面HE染色显示表皮细胞再上皮化加快(P<0.01)、Masson染色显示创面胶原沉积量增加(P<0.01)、CD31免疫荧光染色显示创面血管量增加(P<0.01)。结论年轻化MSC培养基对小鼠创面修复或有促进作用。

Background Mesenchymal stem cell(MSC)conditioned medium without cell therapy is a novel treatment approach in the field of trauma repair.However,its therapeutic efficacy is influenced by the state of MSCs.Objective To explore the effect of rejuvenated mesenchymal stem cell conditioned medium(rejuvenated-MSC-CM)on wound healing in mice.Methods A replicative senescence model of mesenchymal stem cells(MSCs)up to 15-20 passages was established and the indicators of cellular senescence were assessed.Then small molecule compounds,Valproic acid(VPA)and Repsox,were used to reverse the senescence of MSCs and the anti-aging effects were validated.The conditioned medium from senescent MSCs(aging-MSC-CM)and rejuvenated MSCs(rejuvenated-MSC-CM)were collected.Human fibroblasts were cultured with these two conditioned media,and their proliferation,migration,and invasion abilities were evaluated using CCK-8 assay,Ki-67 immunofluorescence,scratch assay,and Transwell assay.In a full-thickness skin defect model on C57BL/6 mice,the wounds were treated with aging-MSC-CM,rejuvenated-MSC-CM,and normal saline,and the effects of these treatments on wound healing were analyzed.Results The expression of SA-β-gal positive cells in senescent MSCs was reduced after treating with small molecules(P<0.01),and immunofluorescence assay showed that the expression levels of senescence genes P21 and P16 were reduced(P21:39.34%±3.72%;P16:17.30%±2.01%;P<0.01).The proliferative,migratory,and invasive capacities of fibroblasts treated with rejuvenated-MSC-CM were enhanced(P<0.01).The C57BL/6 mouse wound model showed increased wound healing in rejuvenated-MSC-CM-treated mice(P<0.01).HE staining of the wounds of mice treated with rejuvenated-MSC-CM showed accelerated re-epithelialization of epidermal cells(P<0.01),Masson staining showed an increase in the amount of collagen deposition on the wounds(P<0.01),and immunofluorescent staining for CD31 showed an increase in the amount of vascularization on the wounds (P<0.01). Conclusion Rejuvenated-MSC-CM may have a promoting effect on wound repair in mice.