双表达外源性4-1BBL/IL-15的K562细胞活化外周血淋巴细胞的研究  

Activation of peripheral blood lymphocytes stimulated by modified K562 cells expressing exogenous 4-1BBL and IL-15

作　　者：曹正锋[1] 万兵[1] 王丽珩[1] 刘丹[1] 龚卫娟[1] 季明春[1] 

机构地区：[1]扬州大学医学院病原生物学和免疫学教研室,扬州225001

出　　处：《现代免疫学》2007年第1期12-17,共6页Current Immunology

基　　金：国家自然科学基金资助项目(30400399;30671917);江苏省自然科学基金资助项目(BK2004404);江苏省高校自然科学基金资助项目(04KJB320162)

摘　　要：为肿瘤过继免疫治疗开发体外激活T细胞、NK细胞的高效途径,研究双表达外源性4-1BBL和IL-15的K562细胞刺激外周血淋巴细胞活化的能力。采用分子克隆技术,分别将4-1BBL和IL-15基因插入双表达载体pVITRO-2,命名为pV4-1BBL-IL-15。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、IL-15分子的K562细胞(K562/4-1BBL/IL-15)。经流式细胞仪(FACS)分选后,K562/4-1BBL/IL15细胞和K562细胞分别用丝裂霉素C处理,与外周血淋巴细胞孵育24 h,FACS检测淋巴细胞表面活化性受体CD69的表达。对NK细胞不仅同时检测活化性受体NKG2D的表达,还用乳酸脱氢酶释放法观察NK细胞受不同刺激细胞作用后,细胞毒活性的变化。结果显示受K562/4-1BBL/IL15细胞刺激后,T细胞CD69的表达无明显变化。γδT细胞CD69表达增长5倍。NK细胞CD69表达增长6倍,而NKG2D的表达增加1.5倍;NK细胞受K562/4-1BBL/IL15细胞作用72 h后,细胞毒活性明显提高。提示双表达4-1BBL/IL-15的K562细胞能够高效激活γδT细胞及NK细胞,有望用于肿瘤的过继免疫治疗。To assess an application of modified K562 cells expressing exogenous 4-1BBL and IL-15 in tumor adoptive immunotherapy, activation of peripheral blood lymphocytes after incubating with the modified K562 cells were investigated in which the 4-1BBL and IL-15 eDNA gene were introduced into respective multi-cloning sites of the double expression eukaryotic vector pVITRO-2, and after identification by DNA sequencing analysis, stable expression of exogenous 4-1BBL and IL-15 in K562 eells were carried out through transfection mediated by liposome. Under the selection of hygromycin B, 4-1BBL expression was eonfirmed by flow cytometery and IL-15 expression by RT-PCR. Sorted by flow cytometery, the modified K562 cells treated by mitomycin C previously were co-cultured with peripheral blood lymphocytes for 24 hours. Expression of activating receptor （CD69） on T cells, γδT cells, and CD69, NKG2D receptors on NK cells were detected by flow cytometery. Cytolysis of NK eells stimulated by K562-4-1BBL-IL-15 cells was detected by LDH method. Results displayed that after co-cultured with K562/ 4-1BBL/IL-15 cells, expression of CD69 had no significant variation compared to stimulation from original K562 cells. However,γδT cells and NK cells up-regulated CD69 expression for 5-folds and 6-folds respectively, the NKG2D expression increased up to 1.5 folds. In addition, eytotoxieity of NK cells stimulated by K562/4-1BBL/IL-15 cells was significantly increased compared to eontrol groups. These results suggested that this kind of modified K562 cells could be used to stimulate γδT and NK eells in vitro for tumor adoptive immunotherapy.

关 键 词：4-1BBL IL-15 K562 表达 活化 

分 类 号：R392.11[医药卫生—免疫学]