供体抗原特异性Treg细胞体外分选与鉴定  

Isolation and identification of recipient CD4^+ CD25^+ Treg cells with donor antigenic specificity in vitro

作　　者：李健[1] 许亚宏[1] 马小平[1] 黄赤兵[2] 张艮甫[2] 

机构地区：[1]解放军452医院泌尿外科,成都610021 [2]第三军医大学附属新桥医院泌尿外科

出　　处：《西南国防医药》2010年第11期1181-1183,共3页Medical Journal of National Defending Forces in Southwest China

基　　金：国家自然科学基金资助项目(30571863)

摘　　要：目的构建大鼠肾移植受体源性CD4+ CD25+ Treg细胞体外分选、鉴定及供体抗原特异性诱导体系。方法大鼠同种异体肾移植受体脾细胞同供肾组织体外混合培养以诱导供体抗原特异性表型;分选受体CD4+ CD25+ T细胞,鉴定CD4+ CD25+ Treg细胞纯度;等量诱导后的Treg细胞加入供、受体大鼠脾细胞混合体系(测定组1)和第三系大鼠同受体大鼠脾细胞混合体系(测定组2)。以受体大鼠脾细胞悬液为阴性对照组,以供、受体大鼠脾细胞混合体系为阳性对照组,检测各组吸光度OD值,观察Treg细胞对两个测定组的抑制效率(IR)。结果 CD4+ CD25+细胞的得率为4.13%,分离纯度为73.34%,CD4+ CD25+ Treg细胞纯度为91.37%。测定组1、2的IR分别为85.4%及21.9%,两者间有显著性差异(P<0.05)。结论体外分选获得高纯度受体源性Treg细胞,并成功诱导获得供体抗原的特异性表型,可满足细胞功能学及在体实验研究。Objective To establish a system for the induction,isolation and identification of CD4＋ CD25＋ regulatory T cells （Treg） with donor antigenic specificity from rat kidney transplant recipients.Methods F344 rats were selected as donors and Lewis rats were selected as recipients in allogenic kidney transplant.Then the splenic cells of Lewis rats were cocultured with the minced kidney tissues of F344 rats for 24 h to induce a phenotype with donor antigenic specificity in vitro.Subsequently,the recipient CD4＋ CD25＋ Treg cells were separated by MACS technology and its purity was identified by fluorescence-activated cell sorting （FACS）.The equal volume of induced Treg cells was respectively added into the splenic cell suspension of F344 plus Lewis rats （group A） and Lewis plus SD rats （group B）.Meanwhile,the splenic cell suspension of Lewis rats was taken as negative control and that of F344 plus Lewis rats was taken as positive control.OD value in each group was determined by MTT method,and then the inhibitory rates （IR） in group A and group B were calculated.Results The yield of CD4＋CD25＋ Treg cells was 4.13% and the purity was 73.34%.The IR in group A and group B was 85.4% and 21.9%,respectively.There was a significant difference between them （P〈0.05）.Conclusion Recipient CD4＋CD25＋ Treg cells with high purity are obtained,and they are successfully induced to have donor antigenic specificity phenotype.These cells can be used for cellular function researches and other studies in vivo.

关 键 词：供体抗原特异性 CD4 CD25 TREG细胞 肾移植 

分 类 号：R318.06[医药卫生—生物医学工程]