γδT细胞在移植排斥反应中具有专职抗原提呈细胞的特性  

Characteristics of gamma-delta T cells as antigen-presenting cells during transplantation rejection

作　　者：张溪[1] 王为忠[1] 董光龙[1] 张洪伟[1] 季刚[1] 

机构地区：[1]解放军第四军医大学西京消化病医院消化外科,陕西省西安市710032

出　　处：《中国组织工程研究与临床康复》2009年第53期10477-10480,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:最新研究表明,γδT淋巴细胞与微生物产物接触后,可表达树突状细胞的特征性标志同时表现出专职抗原提呈细胞的功能,引发CD4+CD8+αβT细胞强烈的免疫应答。目的:初步验证γδT细胞在移植排斥反应中具有专职抗原提呈细胞的特性,摸索一种体外大量扩增γδT细胞的简便方法,并利用基因修饰方法转染FasL至γδT细胞。设计、时间及地点:动物观察实验,于2007-08/2008-08在解放军第四军医大学第一附属医院中心实验室完成。材料:节段性异位小肠移植模型供、受体大鼠分别为清洁级成年健康封闭群雄性Wistar大鼠及SD大鼠,共60只,体质量200～320g。方法:采用三袖套血管吻合法建立大鼠节段性异位小肠移植模型。利用流式细胞仪获取受体大鼠血液中γδT细胞,并初步验证其功能。常规分离获取SD大鼠单个核细胞,以Mtb-Ag进行体外诱导扩增。主要观察指标:观察细胞增殖情况,以TCRγδ磁珠分选试剂盒进行阳性分选,并进行淋巴细胞中γδT细胞所占比例的检测。建立pLXSNFasL反转录病毒转移体系,检测转染后的γδT细胞。结果:活化γδT细胞表现出与树突状细胞相似的细胞黏附功能,在移植排斥反应中γδT细胞具有专职抗原递呈细胞的特性。新鲜分离的单个核细胞中γδT细胞仅占4.5%,Mtb-Ag刺激培养10d后,γδT细胞所占比例可高达72.2%。经免疫磁珠阳性分选,γδT细胞比例高达99.1%。获得的285bpFasL片段,证明PA317细胞中有FasL基因整合。以pLXSNFasL反转录病毒转移体系转染的γδT细胞FasL+细胞占97.3%。结论:实验验证了γδT细胞在移植排斥反应中发挥专职抗原提呈细胞的功能,实现了γδT细胞的体外大量扩增;成功构建了PA317/pLXSN2FasL+病毒上清转染体系,并以Fas配体基因修饰γδT细胞。BACKGROUND： A latest research indicates that γδT cells following touching with microbe products show characteristics as dendritic cells and antigen-presenting cells （APC） and induce intensive immune response of CD4^＋ CD8^ γδT cells. OBJECTIVE： To verify APC-like functions of γδT cells during transplantation rejection, investigate a simple and effective method to amplify γδT cells in vitro, and to infect γδT cells with FasL retrovirus system. DESIGN, TIME AND SETTING： An animal experiment was performed at Central Laboratory of the First Affiliated Hospital of the Fourth Military Medical University of Chinese PLA from August 2007 to August 2008. MATERIALS： A total of 60 healthy adult Wistar rats of clean grade and weighing 200-320 g were used to establish donor and receptor models with segmental heterotopic small intestine transplantation. METHODS： Models of segmental heterotopic small intestine transplantation were established using three sleevelet vascular anastomosis, γδT cells were obtained using flow cytometry and its function was demonstrated, Mononuclear cells were routinely separated and amplified with Mtb-Ag. MAIN OUTCOME MEASURES： Cell proliferation was observed; percentage of γδT cells for lymphocytes was detected using TCR γδ magnetic beads kit; γδT cells following transfection were detected using pLXSN FasL retrovirus system. RESULTS： Activated γδT cells showed dendritic cell-like adhesion function and APC-like functions during transplantation rejection, γδT cells accounted 4.5% for mononuclear cells, the purification of y 6 T cells was up to 72.2% on the 10th day after activated by Mtb-Ag, and the purification of γδT cells was up to 99.1% through positive magnetic sorting. 285 bp FasL fragment demonstrated that the gene integration was observed in PA317 cells. The ration of FasL＋ cells was 97.3% after infected by pLXSN FasL retrovirus system. CONCLUSION： This study demonstrated APC-like functions of γδT cells during transplantation rejection. γδT cel

关 键 词：ΓΔT细胞 抗原提呈细胞 基因修饰 大鼠 小肠移植 

分 类 号：R617[医药卫生—外科学]