固有淋巴细胞3在皮肤创伤愈合中的作用及功能研究  

Role of group 3 innate lymphoid cells in skin wound healing and underlying mechanisms

作　　者：张威 母兴语 杭倩如 黄诒洁 徐腾姣 何小解[3] 丁艳 Zhang Wei;Mu Xingyu;Hang Qianru;Huang Yijie;Xu Tengjiao;He Xiaojie;Ding Yan(Department of Dermatology,Affiliated Dermatology Hospital of Hainan Medical University,Haikou 570100,China;The First Clinical College of Hainan Medical University,Haikou 570199,China;Children's Medical Center,the Second Xiangya Hospital of Central South University,Changsha 410011,China)

机构地区：[1]海南医学院附属皮肤病医院皮肤科,海口570100 [2]海南医学院第一临床学院,海口570199 [3]中南大学湘雅二医院儿童医学中心,长沙410011

出　　处：《中华皮肤科杂志》2024年第6期516-523,共8页Chinese Journal of Dermatology

基　　金：海南省重点研发计划项目(ZDYF2020147);海南省临床医学中心建设项目(QRCBT202121)。

摘　　要：目的探讨固有淋巴细胞3(ILC3)在皮肤创伤愈合中的作用机制。方法24只5周龄C57BL/6雄性小鼠随机分为皮肤创伤+ILC3抑制剂组(简称ILC3抑制剂组)、皮肤创伤组和对照组,每组8只;建立创伤模型前4 d给予ILC3抑制剂组小鼠腹腔注射1μg ILC3抑制剂每2天1次共2次,皮肤创伤组注射等体积生理氯化钠溶液,对照组小鼠正常喂养。构建小鼠皮肤创伤模型:腹腔注射麻醉后,以背部中线中点为圆点,用环钻压切出直径0.6 cm的皮肤全层圆形切口,并经组织学证明为全层损伤。观察记录创面大小,分别于创伤第0、1、3、5、7、9天对创面拍照并计算相应的创面愈合率。术后第9天取创缘组织用流式细胞仪分析皮肤创周ILC3浸润数量,并行HE染色,观察创面皮肤组织愈合情况,qRT-PCR检测创缘组织维生素D受体(VDR)、Notch1、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-17A、IL-17F、IL-22 mRNA表达水平,Western印迹法检测其蛋白表达水平。组间比较采用单因素方差分析及t检验。结果创伤后第9天,与对照组比较,皮肤创伤组创缘组织ILC3数量升高(5.31%±1.47%比3.10%±0.54%,P<0.01),TNF-α、IL-22、IL-17A、IL-17F mRNA及蛋白表达水平较高(均P<0.05),VDR mRNA及蛋白表达水平较低(P<0.05),Notch1蛋白表达水平较高(P<0.05),但其mRNA表达水平两组间差异无统计学意义(P>0.05)。第1、3、5天,ILC3抑制剂组创面愈合率(45.17%±9.90%、61.58%±11.61%、75.61%±9.12%)均高于皮肤创伤组(25.87%±10.96%、47.78%±13.81%、64.55%±10.29%,均P<0.05),第9天创周ILC3数量(2.69%±0.95%)低于皮肤创伤组(P<0.01),创缘组织TNF-α、IL-22、IL-17A、IL-17F mRNA及蛋白表达均低于皮肤创伤组(均P<0.05),Notch1、VDR mRNA及蛋白表达均高于皮肤创伤组(均P<0.05)。创伤后第9天组织病理检查显示,ILC3抑制剂组上皮结构连续、完整,胶原纤维更加密集且排列整齐,毛囊、血管、皮脂腺等各组织结构更接近对照组皮肤。结�Objective To investigate the role of group 3 innate lymphoid cells(ILC3)in skin wound healing,and to explore the underlying mechanisms.Methods Twenty-four 5-week-old male C57BL/6 mice were randomly and equally allocated into 3 groups:the skin wound+ILC3 inhibitor group(referred to as ILC3 inhibitor group),the skin wound group,and the control group,with 8 mice in each group.Four days before the establishment of the wound model,mice in the ILC3 inhibitor group were intraperitoneally injected with 1μg of ILC3 inhibitor every 2 days for a total of 2 doses,mice in the skin wound group were injected with an equal volume of physiological saline solution,and mice in the control group were fed normally.To establish a mouse skin wound model,a full-thickness circular incision with a diameter of 0.6 cm was made around the midpoint of the dorsal midline using a biopsy punch after the intraperitoneal injection of anesthetics,which was histologically confirmed to be a full-thickness injury.The size of the wounds was observed and recorded,photographs of the wounds were taken on days 0,1,3,5,7,and 9 after wounding,and corresponding wound healing rates were calculated.On day 9 after wounding,tissue samples were collected from the wound edges,and subjected to flow cytometry analysis to quantify ILC3 infiltrating around the skin wound,and hematoxylin and eosin(HE)staining was performed to assess the healing status of the skin wounds.Real-time quantitative polymerase chain reaction(qRT-PCR)was conducted to determine the mRNA expression of vitamin D receptor(VDR),Notch1,tumor necrosis factor-alpha(TNF-α),interleukin(IL)-17A,IL-17F,and IL-22 in the wound-edge tissues,and Western blot analysis to determine their protein expression.Statistical analysis was carried out by using one-way analysis of variance and t test.Results On day 9 after wounding,the skin wound group showed an increased number of ILC3 in the wound-edge tissues(5.31%±1.47%vs.3.10%±0.54%,P<0.01),increased mRNA and protein expression of TNF-α,IL-22,IL-17A,and IL-17F(a

关 键 词：淋巴细胞 伤口愈合 白细胞介素类 肿瘤坏死因子Α 受体 骨化三醇 受体 Notch1 固有淋巴细胞 

分 类 号：R751[医药卫生—皮肤病学与性病学]