双表达外源性4-1BBL/RANTES的K562细胞活化外周血淋巴细胞的研究

目的探讨K562细胞表达外源性蛋白4-1BBL/RANTES后,对T细胞、NK细胞有无活化作用,为进一步构建人工抗原提呈细胞提供前提。方法采用分子克隆技术,分别将4-1BBL和RANTES基因插入双表达载体pVITRO-2,命名为pV4-1BBL-RANTES。经测序鉴定后,利用脂质体介导的转染及潮霉素筛选,获得稳定双表达4-1BBL、RANTES分子的K562细胞(K562/4-1BBL/RANTES)。经流式细胞仪(FACS)分选后,K562/4-1BBL/RANTES细胞用丝裂霉素C处理,与外周血淋巴细胞孵育24,FACS检测淋巴细胞表面活化性受体CD69的表达;对NK细胞同时检测了活化性受体NKG2D的表达情况。结果受K562/4-1BBL/RANTES细胞刺激后,CD3+T细胞、CD4+T细胞、γδT细胞和NK细胞的活化性受体CD69的表达与未受刺激前相比,均有明显上调;但与单纯K562细胞刺激组相比,无显著差异。另外,CD8+T细胞CD69的表达及NK细胞NKG2D的表达无明显变化。结论将4-1BBL和RANTES共表达于K562细胞,不具备活化淋巴细胞的效应。

Activation of Peripheral Blood Lymphocytes Stimulated by Modified K562 Cells Expressing Exogenous 4-1BBL and RANTES

Objective To observe whether modified K562 cells double expressing exogenous 4-1BBL and RANTES can stimulate peripheral blood lymphocytes.Methods Human 4-1bbl and RANTES cDNA gene were introduced into respective multi-cloning sites of the double expression eukaryotic vector-pVITRO-2 with the recombinant DNA technique.After identification by DNA sequencing analysis,stable expression of exogenous 4-1BBL and RANTES in K562 cells were carried out through transfection mediated by liposome.Under the selection of hygromycin B,4-1BBL expression was confirmed by flow cytometery and RANTES expression by RT-PCR.After being sorted by flow cytometery,the modified K562 cells were treated by mitomycin C before co-cultured with peripheral blood lymphocytes for about 24 hours.Expression of activating receptor(CD69) on T cells,γδT cells,and CD69,NKG2D receptors on NK cells were detected by flow cytometery.Results After having being co-cultured with K562/4-1BBL/RANTES cells,CD3+T cell、CD4+T cell、γδT cell and NK cell had up-regulated CD69 expression.However,there were not significant differences of expressi on of CD69 on the surface of above lymphoctes in comparison with stimula-tion by original K562 cells.Furthermore,expression of CD69 on CD8+ T cells and NKG2D on NK cell had no significant variation compared with no-stimulation and stimulation from original K562 cells.Conclusions Modified K562 cells with co-expression of 4-1BBL and RANTES could not activate T cell and NK cell.