摘要
目的 探讨淫羊藿苷(icariin,ICA)增强脂肪干细胞(adipose-derived stem cells,ADSCs)促进大鼠皮肤放射性损伤修复的疗效及初步机制。方法 取12只SPF级SD大鼠,体质量(220±10)g,采用X线照射背部1.5 cm×1.5 cm皮肤,单次剂量10 Gy,每次照射剂量率200 c Gy/min,制备皮肤放射性损伤模型。造模成功后随机分为4组,每组3只,第2天在照射部位创面皮下注射相应细胞:A组为1×10^(7)个/m L大鼠ADSCs 0.1 m L,B组为1×10^(7)个/m L大鼠ADSCs 0.1 m L+1μmol/L ICA 0.1 m L,C组为缺氧诱导因子2α(hypoxia-inducible factor 2α,HIF-2α)抑制剂(-)处理后的1×10^(7)个/m L大鼠ADSCs 0.1 m L+1μmol/L ICA 0.1 m L,D组为Notch1抑制剂(-)处理后的1×10^(7)个/m L大鼠ADSCs 0.1 m L+1μmol/L ICA 0.1 m L;均为单次给药。造模后1~7 d连续观察大鼠背部辐照区皮肤损伤情况;造模后28 d处死各组大鼠,取背部辐照区皮肤组织,行HE染色和Masson染色观察皮肤修复状况并定量检测胶原纤维含量,免疫组织化学染色检测CD31表达量,Western blot和实时荧光定量PCR(real-time fluorescence quantitative PCR,q RT-PCR)分别检测VEGF、PDGF-BB、FGF-2、IL-10、TGF-β、HIF-2α及Notch1、2、3蛋白和m RNA相对表达量。结果 造模后各组大鼠均出现皮肤溃疡、红肿等情况;3 d时各组均有组织液渗出;7 d时可观察到B组皮肤损伤范围明显小于其余3组。造模后28 d,A组表皮层增厚,真皮层纤维排列略紊乱,可见少量炎症细胞聚集;B组表皮层趋于正常,真皮层纤维排列趋于整齐,无明显炎症细胞聚集,新生血管增多;C、D组表皮层厚度增加,真皮层纤维排列紊乱且存在断裂现象。B组胶原纤维含量显著高于其余3组,D组低于A组,差异均有统计学意义(P<0.05)。免疫组织化学染色示B组CD31表达量显著高于其余3组,C、D组低于A组,差异均有统计学意义(P<0.05)。Western blot和q RT-PCR检测示,B组VEGF、PDGF-BB、FGF-2、IL-10、TGF-β、HIF-2α及Notch1、2、3蛋白及m RNA相对表达量显著高于其余3组,差异均有统计学意义(P<0.05)。结论 ICA可能通过HIF-2α-VEGF-Notch通路增强ADSCs促进血管生成、减轻炎症反应,有利于大鼠皮肤放射性损伤修复。
Objective To investigate the effectiveness and preliminary mechanisms of icariin(ICA)in enhancing the reparative effects of adipose-derived stem cells(ADSCs)on skin radiation damagies in rats.Methods Twelve SPFgrade Sprague Dawley rats[body weight(220±10)g]were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin,with a dose rate of 200 cGy/min to make skin radiation damage model.After successful modelling,the rats were randomly divided into 4 groups(n=3),and on day 2,the corresponding cells were injected subcutaneously into the irradiated wounds:group A received 0.1 mL of rat ADSCs(1×10^(7)cells/mL),group B received 0.1 mL of rat ADSCs(1×10^(7)cells/mL)+1μmol/L ICA(0.1 mL),group C received 0.1 mL of rat ADSCs(1×10^(7)cells/mL)pretreated with a hypoxia-inducible factor 2α(HIF-2α)inhibitor+1μmol/L ICA(0.1 mL),and group D received 0.1 mL of rat ADSCs(1×10^(7)cells/mL)pretreated with a Notch1 inhibitor+1μmol/L ICA(0.1 mL).All treatments were administered as single doses.The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling.On day 28,the rats were sacrificed,and skin tissues from the irradiated areas were harvested for histological examination(HE staining and Masson staining)to assess the repair status and for quantitative collagen content detection.Immunohistochemical staining was performed to detect CD31 expression,while Western blot and real-time fluorescence quantitative PCR(qRT-PCR)were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor(VEGF),platelet-derived growth factor BB(PDGF-BB),fibroblast growth factor 2(FGF-2),interleukin 10(IL-10),transforming growth factorβ(TGF-β),HIF-2α,and Notch1,2,and 3.Results All groups exhibited skin ulcers and redness after irradiation.On day 3,exudation of tissue fluid was observed in all groups.On day 7,group B showed significantly smaller skin injury areas compared to the other 3 groups.On day 28,histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A.In group B,the epidermis appeared more normal,the dermal fibers were more orderly,and there was an increase in new blood vessels without significant inflammatory cell aggregation.In contrast,groups C and D showed significantly increased epidermal thickness,disordered and disrupted dermal fibers.Group B had higher collagen fiber content than the other 3 groups,and group D had lower content than group A,with significant differences(P<0.05).Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups,while groups C and D had lower expression than group A,with significant differences(P<0.05).Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF,PDGF-BB,FGF-2,IL-10,TGF-β,HIF-2α,and Notch1,2,and 3 proteins and mRNAs compared to the other 3 groups(P<0.05).Conclusion ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
作者
左彧儿
黎双溢
谭斯予
胡啸昊
李骤
李浩曦
ZUO Yuer;LI Shuangyi;TAN Siyu;HU Xiaohao;LI Zhou;LI Haoxi(Department of Plastic and Reconstructive Surgery,Guangxi Academy of Medical Sciences&People’s Hospital of Guangxi Zhuang Autonomous Region,Nanning Guangxi,530021,P.R.China;Liaoning University of Traditional Chinese Medicine Xinglin College,Shenyang Liaoning,110000,P.R.China;Department of Radiation Oncology,the First People’s Hospital of Nanning,Nanning Guangxi,530022,P.R.China;Department of Spine Surgery,Guangxi Academy of Medical Sciences&People’s Hospital of Guangxi Zhuang Autonomous Region,Nanning Guangxi,530021,P.R.China)
出处
《中国修复重建外科杂志》
北大核心
2025年第7期881-890,共10页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金项目(82460449)
广西自然科学基金项目(2024GXNSFAA010169)
南宁市优秀青年科技创新创业人才培育项目(20220108)
广西再生医学重点实验室开放课题(桂再重开202007)
广西科技基地和人才专项(桂科AD21220134)。
