Vγ4T细胞在应用雷帕霉素的小鼠全层皮肤缺损创面愈合障碍中的作用及其机制  

Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice

作　　者：刘中阳[1] 程旭 张景霞 张建文[1] 郭丽丽[1] 李广帅[1] 史珂 Liu Zhongyang;Cheng Xu;Zhang Jingxia;Zhang Jianwen;Guo Lili;Li Guangshuai;Shi Ke(Medical Cosmetic Center,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院医学美容中心,郑州450052

出　　处：《中华烧伤与创面修复杂志》2022年第5期462-470,共9页Chinese Journal of Burns And Wounds

基　　金：郑州大学第一附属医院青年基金项目。

摘　　要：目的探讨Vγ4T细胞在应用雷帕霉素的小鼠全层皮肤缺损创面愈合障碍中的作用及其机制。方法采用实验研究方法,选取86只8~12周龄雄性C57BL/6J小鼠(以下简称野生型小鼠)进行后续实验。取5只野生型小鼠,从其腋窝淋巴结分离Vγ4T细胞用于后续实验。取42只野生型小鼠,腹腔注射雷帕霉素建立应用雷帕霉素的小鼠模型,用于后续实验。取18只野生型小鼠,按随机数字表法(分组方法下同)分为不进行任何处理的正常对照组和单纯创伤组、创伤+CC趋化因子配体20(CCL20)抑制剂组(每组6只),将后2组小鼠背部制成全层皮肤缺损创面(创面模型下同),创伤+CCL20抑制剂组小鼠伤后连续3 d于创缘皮下注射CCL20抑制剂,另取6只应用雷帕霉素的小鼠建立创面模型作为雷帕霉素+创伤组,伤后3 d,采用酶消化法提取各创伤小鼠创周皮肤组织的表皮细胞,采用流式细胞仪检测表皮细胞中Vγ4T细胞的百分比。于适宜时间点取正常对照组小鼠背部正常皮肤组织的表皮细胞同前进行检测。取5只野生型小鼠建立创面模型,伤后3 d,提取创周皮肤组织的表皮细胞,采用流式细胞分选仪将细胞群分为Vγ4T细胞、Vγ3T细胞及γδ阴性细胞,分别设为Vγ4T细胞组、Vγ3T细胞组及γδ阴性细胞组(均与B16小鼠黑色素瘤细胞混合),以单纯B16小鼠黑色素瘤细胞为黑色素瘤细胞对照组,采用实时荧光定量反转录PCR(RT-PCR)法检测各组细胞白细胞介素22(IL-22)mRNA表达情况(样本数为6)。取30只应用雷帕霉素的小鼠建立创面模型,伤后即刻分为进行相应注射处理的单纯Vγ4T细胞组与Vγ4T细胞+IL-22抑制剂组以及注射PBS的雷帕霉素对照组(每组10只);另取10只野生型小鼠建立创面模型并注射PBS作为野生型对照组。各组小鼠均连续注射6 d,伤后1、2、3、4、5、6 d于当日注射后计算4组小鼠创面面积百分比。分别取6只野生型小鼠和6只应用雷帕霉素Objective To investigate the role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice.Methods The experimental research methods were applied.Eighty-six C57BL/6J male mice(hereinafter briefly referred to as wild-type mice)aged 8-12 weeks were selected for the following experiments.Vγ4 T cells were isolated from axillary lymph nodes of five wild-type mice for the following experiments.Intraperitoneal injection of rapamycin for 42 mice was performed to establish rapamycin-treated mice model for the following experiments.Eighteen wild-type mice were divided into normal control group without any treatment,trauma only group,and trauma+CC chemokine ligand 20(CCL20)inhibitor group according to the random number table(the same grouping method below),with 6 mice in each group.The full-thickness skin defect wound was made on the back of mice in the latter two groups(the same wound model below),and mice in trauma+CCL20 inhibitor group were continuously injected subcutaneously with CCL20 inhibitor at the wound edge for 3 days after injury.Another 6 rapamycin-treated mice were used to establish wound model as rapamycin+trauma group.On post injury day(PID)3,the epidermal cells of the skin tissue around the wound of each trauma mice were extracted by enzyme digestion,and the percentage of Vγ4 T cells in the epidermal cells was detected by flow cytometry.In normal control group,the epidermal cells of the normal skin tissue in the back of mice were taken at the appropriate time point for detection as above.Five wild-type mice were used to establish wound models.On PID 3,the epidermal cells were extracted from the skin tissue around the wound.The cell populations were divided into Vγ4 T cells,Vγ3 T cells,andγδnegative cells by fluorescence-activated cell sorter,which were set as Vγ4 T cell group,Vγ3 T cell group,andγδnegative cell group(with cells in each group being mixed with B16 mouse melanoma cells),respectively.B16 mouse melanoma cells were used as melanoma

关 键 词：西罗莫司 伤口愈合 趋化因子CCL20 Vγ4T细胞 白细胞介素22 

分 类 号：R641[医药卫生—外科学]