中国药理学与毒理学杂志2025，Vol.39Issue(12)：913-922,10.DOI:10.3867/j.issn.1000-3002.2025.08596
间充质干细胞衰老削弱其条件培养基促小鼠皮肤伤口愈合作用及机制
Cellular senescence diminishes wound-healing-promoting effects of mesenchymal stem cell-conditioned medium
摘要
Abstract
OBJECTIVE To investigate the impact of cellular senescence on the wound healing-promoting effect of mesenchymal stem cell-conditioned medium(MSC-CM)and explore the underlying mechanisms in order to provide data for the clinical applications of stem cell therapy.METHODS An MSC senescence model was established via induction with etoposide(ETO).Five concentration gradi-ents of ETO 0,1,5,10 and 20 μmol·L-1 were set,and MSCs were treated for 24 h or 48 h.After treat-ment,cell proliferation was detected using the cell counting kit-8(CCK-8)assay,the proportion of senescent cells was determined by β-galactosidase staining,and the mRNA expression levels of senescence-related genes—tumor protein P53(TP53),cyclin-dependent kinase inhibitor 1A(Cdkn1a),and cyclin-dependent kinase inhibitor 2A(Cdkn2a)—were measured by RT-qPCR.After the optimal condition for cell senescence induction was established,the corresponding conditioned medium was collected for subsequent animal experiments.Full-thickness skin wounds were induced in male Kunming mice that were randomized into 3 groups:the MSC-CM group(conditioned medium from normal MSCs),ETO-CM group(conditioned medium from senescent MSCs),or control(DMEM complete medium).All conditioned media were harvested after 24 h of culture.Each wound was given 20 μL of the respec-tive treatment twice per day(in the morning and evening).On post-modeling days 0,1,3,5,9 and 14,the wound sites of mice were photographed to calculate the wound closure rates.Wound tissues collected on days 3,5,9 and 14 were processed for HE staining to evaluate the effects of different treatments on wound healing.RT-qPCR was performed on day 3 on wound tissues to measure the expression levels of inflammatory cytokines[inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β,IL-10 and transforming growth factor-β(TGF-β)].Immuno-histochemistry was performed to detect the expressions of inflammatory factors IL-6 and IL-10 in wound tissues on post-modeling day 3,and the expression of angiogenesis factor CD31 on post-model-ing day 5.Masson staining was used to evaluate the collagen fiber content in wound tissues on post-modeling day 9.RESULTS Compared to the ETO 0 μmol·L-1 group,24 h induction with 5 μmol·L-1 ETO had no effect on cell proliferation,but significantly increased the proportion of β-galactosidase-positive cells and up-regulated the mRNA expression levels of TP53,Cdkn1a,and Cdkn2a.Compared to the ETO-CM group,MSC-CM treatment significantly enhanced skin wound healing,as evidenced by increased wound closure rates,greater neo-epithelial thickness,and accelerated re-epithelialization.On post-modeling day 3,MSC-CM treatment significantly downregulated mRNA expressions of the pro-inflammatory cytokine IL-6 while upregulating anti-inflammatory IL-10 in wound tissues compared with the control group.Compared with MSC-CM treatment,ETO-CM treatment promoted the expressions of pro-inflammatory cytokines(iNOS,IL-6,and IL-1β).Compared to the control and ETO-CM groups,the MSC-CM group significantly reduced the protein expression of the pro-inflammatory cytokine IL-6 in wounds.No significant differences were observed in collagen deposition or angiogenesis between the control,MSC-CM,and ETO-CM groups.CONCLUSION MSC senescence can attenuate the therapeutic effects of MSC-CM on skin wound healing,potentially by abrogating MSC-mediated regulation of wound inflammatory cytokines.关键词
间充质干细胞条件培养基/干细胞治疗/细胞衰老/伤口愈合/炎症因子/皮肤损伤Key words
mesenchymal stem cell-conditioned medium/stem cell therapy/cellular senescence/wound healing/inflammatory cytokines/skin injury分类
医药卫生引用本文复制引用
梁舒婷,杨雪舟,张博文,张亚平,梁桃缘,苟若兮,王东恩,续惠云..间充质干细胞衰老削弱其条件培养基促小鼠皮肤伤口愈合作用及机制[J].中国药理学与毒理学杂志,2025,39(12):913-922,10.基金项目
国家自然科学基金(32471510) National Natural Science Foundation of China(32471510) （32471510）
