γδT细胞在移植排斥反应中具有专职抗原提呈细胞的特性

背景:最新研究表明,γ~δT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γ~δT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γ~δT细胞的简便方法,并利用基因修饰方法转染FasL 至γ~δT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200～320 g.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γ~δT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TCRγ~δ磁珠分选试剂盒进行阳性分选,并进行淋巴细胞中γ~δT细胞所占比例的检测.建立pLXSN FasL反转录病毒转移体系,检测转染后的γ~δT细胞.结果:活化γ~δT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γ~δT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γ~δT 细胞仅占4.5%,Mtb-Ag 刺激培养10 d 后,γ~δT 细胞所占比例可高达72.2%.经免疫磁珠阳性分选,γ~δT 细胞比例高达99.1%.获得的285 bp FasL片段,证明PA317细胞中有FasL基因整合.以pLXSN FasL反转录病毒转移体系转染的γ~δT细胞FasL~+细胞占97.3%.结论:实验验证了γ~δT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γ~δT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γ~δT细胞.

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

BACKGROUND: A latest research indicates that γ~δ T cells following touching with microbe products show characteristics as dendritic cells and antigen-presenting cells (APC) and induce intensive immune response of CD4~+ CD8~+ γ~δ T cells.OBJECTIVE: To verify APC-like functions of γ~δ T cells during transplantation rejection, investigate a simple and effective method to amplify γ~δ T cells in vitro, and to infect γ~δ T 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. γ~δ T 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 γ~δ T cells for lymphocytes was detected using TCRγ~δ magnetic beads kit; γ~δ T cells following transfection were detected using pLXSN FasL retrovirus system. RESULTS: Activated γ~δ T cells showed dendritic cell-like adhesion function and APC-like functions during transplantation rejection. γ~δ T cells accounted 4.5% for mononuclear cells, the purification of γ~δ T cells was up to 72.2% on the 10~(th) day after activated by Mtb-Ag, and the purification of γ~δ T 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 γ~δ T cells during transplantation rejection. γ~δ T cells were successfully amplified in vitro. PA317/ pLXSN2FasL+ retrovirus system was successfully constructed and γ~δ T cells were modified by FasL retrovirus system.