异种HSP_(70)及固相抗体法体外扩增人γδT细胞的比较

Comparison of expansion of human γδT lymphocytes with solid phase antibody,HSP_(70BCG) and HSP_(70m)-peptide complexes in vitro

目的 :比较异种HSP70 及固相抗体法体外扩增人γδT细胞。方法 :分别用固相抗体法、HSP70BCG 及小鼠榄香烯复合瘤苗热休克蛋白 70 肽复合物 (HSP70m 肽复合物 )体外扩增人PBMCs的γδT细胞 ,流式细胞仪检测细胞表型及亚型分布 ,RT PCR检测各亚型基因的取用。MTT检测γδT细胞的细胞毒作用。结果 :培养 14天后 ,抗体法扩增的PMBCs总数增加 30～ 4 0倍 ,γδT细胞的比率由 3.2 %提高到 86 .7% ;HSP70BCG 组PBMCs总数增加 7～ 8倍 ,γδT细胞的比率由 3.2 %提高到 71.32 % ;HSP70m 肽复合物组PBMCs总数增加 4～ 5倍 ,γδT细胞的比率由 3.2 %提高到 2 7.2 6 %。抗体及HSP70BCG 扩增的γδT细胞以表达Vγ9 Vδ2亚型为主 ,且Vδ1、Vδ2、Vδ3三种基因均得到取用 ,对Daudi具较高的杀伤活性。结论 :抗体及HSP70BCG 均能扩增出高纯度的γδT细胞 ,且具有完整的受体库 ,扩增的γδT细胞具细胞毒活性。

Objective:To compare the human γδT lymphocytes expanded with HSP 70BCG and HSP 70 m-peptide complexes with those expanded with solid phase antibody in vitro.Methods:PBMCs were cultured with anti-TCR γδ antibody?HSP 70BCG and HSP 70 m -peptide complexes.The total cell number was counted.The flow cytometer was used to analyze the lymphocytes phenotypes and subtypes.RT-PCR was used to analyze the level of Vδ mRNA.The cytotoxitic activity of γδT cells against Daudi was determined using MTT calorimetric assay.Results:After 14 days of culture,in the antibody culture system,the total cell number increased about 30-40 fold,and the ratio of γδT cell reached to 86.3%;In the HSP 70BCG culture system,the total cell number increased about 7-8 fold,and the ratio of γδT cell reached to 71.23%;In the HSP 70-peptide complexes culture system,the total cell number increased about 4-5 fold,and the ratio of γδT cells reached to 27.26%.The antibody and HSP 70BCG activated γδT cells possessed a whole repertoire and mainly expressed Vγ9/Vδ2 subset and exhibited a strong cytotoxicity against Daudi.Conclusion:Anti-TCR γδ antibody and HSP 70BCG were able to proliferate purer γδT cells.The cells had a whole repertoire,and exhibited strong cytotoxicity against Daudi.