树突状表皮T淋巴细胞在小鼠皮肤移植免疫排斥反应中的作用及机制  

Role of dentritic epidermal T lymphocytes in immune rejection of skin allograft in mice and its mech-anism

作　　者：黄华[1] 燕荣帅[1] 刘美希[1] 周俊峄[1] 谭江琳[1] 张晓容[1] 胡晓红[1] 黄勇[1] 贺伟峰[1] 吴军[1] 罗高兴[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆市疾病蛋白质组学重点实验室,重庆400038

出　　处：《中华烧伤杂志》2015年第2期125-129,共5页Chinese Journal of Burns

基　　金：国家自然科学基金（81372082、81373155）

摘　　要：目的探讨树突状表皮T淋巴细胞（DETC）在小鼠皮肤移植免疫排斥反应中的作用及其相天机制。方法（1）取1只野生型C57BL／6雄性小鼠背部全层皮肤，分离表皮细胞，流式细胞仪检测DETC表达情况及细胞表型。另取1只野生型C57BL／6雄性小鼠背部全层皮肤，分离表皮，免疫荧光技术观察DETC形态学特征。（2）分别取4只绿色荧光蛋白（GFP）标记C57BL／6雄性小鼠、7只野生型C57BL／6雌性小鼠（野生型组）以及7只γδ8T淋巴细胞6基因敲除C57BL／6雌性小鼠（基因敲除组）。剪去野生型组与基因敲除组小鼠背部1．4cm×1．4cm大小全层皮肤，移植GFP标记C57BL／6雄悱小鼠背部1．2cm×1．2cm大小全层皮肤，通过小动物活体成像仪及肉眼观察，判定并记录移植皮片存活时间。（3）取2只野生型C57BL／6雄性小鼠，分离表皮细胞，加入48孔板，按随机数字表法分为激活组和对照组，每组4孔。激活组加入2g／mL刀豆蛋白A10μL，对照组加入等量PBS，处理24h后用流式细胞仪枪测DETC表达γ干扰素情况。（4）取4只GFP标记C57BL／6雄性小鼠（供体）；取14只野生型C57BL／6雌性小鼠（受体），按随机数字表法分为γ干扰素中和组和对照组，每组7只。按（2）中方法构建皮肤移植模型，术前和术后72h，γ干扰素中和组小鼠腹腔注射1mg／mLγ干扰素中和抗体200μL，对照组注射等量生理盐水，同（2）中方法观察并记录移植皮片存活时间，并将γ干扰素中和组与（2）中基因敲除组移植皮片存活时间进行比较。对皮片生存曲线进行Log-rank（Mantel-Cox）检验。结果（1）小鼠皮肤表皮细胞内DETC阳性表达率为7．27％，且DETC均为CD3^＋表型细胞。DETC呈树突状散在分布于小鼠皮肤表皮中。（2）基因敲除组小鼠移植皮片存活时间为22～35d，较野生型组的12～16d明显延长（χ^2=14．10，P〈0．001），（3）激活组DEObjective To explore the role of dentritic epidermal T lymphocytes （DETCs） in im-inune rejeetion of skin allograft in mice and its related mechanism. Methods （ 1 ） Full-thickness skin was harvested from back of one male wild type （WT） C57BL/6 mouse. Epithelial cells were isolated for detee- tiou of the expression of DETCs and their phenotype with flow cytometer. Another male WT C57BL/6 mouse was used to harvest full-thickness skin from the back. Epidermis was isolated for observation of the morpho- logical characleristics of DETCs with immunofluoreseenee technology. （2） Four male green fluoreseenee pro- tein （（,FP） -marked C57B1,/6 mice, 7 female WT C57BL/6 mice （ group WT） , and 7 female γδST lympho-cytes g gene knock-out （GK） C57BL/6 mice （group GK） were used. Full-thickness skin in the size of 1.4 cm × 1. 4 cm on the back of mice in groups WT and GK were excised, and the wounds were transplanted with full-thickness skin in the size of 1.2 cm × 1.2 cm obtained from male GFP-marked C57BL/6 mice. The survival time of skin grafts was affirmed with small animal in vivo imager and naked eyes and recorded. （3） Two male WT C57BL/6 mice were used to isolate epithelial ceils. Cells were inoculated into 48-well plate and divided into activation group （A） and control group （C） according to the random number table, with 4 wells in each group. Cells in group A were treated with 10 μL concanavalin A in the concentration of 2μg/mL for 24 hours, while those in group C with PBS in the same volume as that in group A. The expres- sion of interferon γ in DETCs was detected with flow cytnmeter. （4） Four male GFP-marked C57BL/6 mice were used as donors. Fourteen female WT C57BL/6 mice were used as receptors and divided into interferon γ neutralizing group （IN） and control group （C） according to the random number table, with 7 mice in each group. The skin transplantation model of C57BL/6 male to C57BL/6 female was established as in part （2）. Before surgery and 72 h

关 键 词：皮肤移植 干扰素Γ 移植免疫学 树突状表皮T淋巴细胞 

分 类 号：R622[医药卫生—整形外科]