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关机制. 方法 (1)取1只野生型C57B L/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γδT淋巴细胞δ基因敲除C57B L/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1.4 cm×1.4 cm大小全层皮肤,移植GFP标记C57BL/6雄性小鼠背部1.2 cm×1.2 cm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C57B L/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2 μg/mL刀豆蛋白A 10 μL,对照组加入等量PBS,处理24 h后用流式细胞仪检测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C57 BL/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72 h,γ干扰素中和组小鼠腹腔注射1 mg/mLγ干扰素中和抗体200 μL,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 ～ 35 d,较野生型组的12 ～ 16 d明显延长(x2=14.10,P＜0.001).(3)激活组DETC的γ干扰素表达率为22.70％,明显高于对照组的0.51％.(4)γ干扰素中和组小鼠移植皮片存活时间为19 ～24 d,较对照组的12～16 d明显延长(x2=13.60,P＜0.001),但与(2)中基因敲除组小鼠移植皮片存活时间相近(x 2 =0.06,P=0.810). 结论 DETC在C57BL/6小鼠皮肤移植免疫排斥反应中起促进作用,该作用可能是通过其分泌γ干扰索实现的.

abstractsObjective To explore the role of dentritic epidermal T lymphocytes (DETCs) in immune rejection 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 detection 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 morphological characteristics of DETCs with immunofluorescence technology.(2) Four male green fluorescence protein (GFP)-marked C57BL/6 mice,7 female WT C57BL/6 mice (group WT),and 7 female γδT lymphocytes δ 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 cells.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 2 μg/mL for 24 hours,while those in group C with PBS in the same volume as that in group A.The expression of interferon γ in DETCs was detected with flow cytometer.(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 hours after,mice in group IN were intraperitoneally injected with 200 μL interferon γ neutralizing antibody in the concentration of 1 mg/mL,and those in group C with normal saline in the same volume as that in group IN.The survival time of skin grafts was observed and recorded using the methods in part (2),and the result of group IN was compared with that of group GK in part (2).The survival curve of skin grafts was processed with Log-rank (Mantel-Cox) test.Results (1) The positive expression rate of DETCs in epithelial cells of skin in mouse was 7.27％,and they were all CD3 + cells.DETCs were found to be scattered in the epidermis of skin in mouse with dendritic morphology.(2) The survival time of skin gratts of mice in group GK was 22-35 d,obviously longer than that in group WT (12-16 d,x2 =14.10,P ＜ 0.001).(3) Expression of interferon γ was detected in 22.70％ DETCs in group A,which was obviously higher than that in group C (0.51％).(4) The survival time of skin grafts of mice in group IN was 19-24 d,which was obviously longer than that in group C (12-16 d,x2 =13.60,P ＜ 0.001) but close to that in group GK as in part (2) (x2 =0.06,P =0.810).Conclusions DETCs are involved in promotion of immune rejection of skin allograft probably by secreting interferon γ.