不同遗传背景NK细胞对转化细胞的杀伤效应  

Natural killer cells from different genetic backgrounds exert cytotoxic effects on transformed cells

作　　者：王杨[1] 郭坤元[1] 黄宇贤[1] 周健[1] 

机构地区：[1]南方医科大学珠江医院血液科,广东省广州市510280

出　　处：《中国组织工程研究与临床康复》2008年第18期3471-3474,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30471636)~~

摘　　要：目的:细胞免疫治疗的着眼点不仅是杀伤局部异常生长的肿瘤细胞,还要应对潜在转化细胞的影响,为此观察3种不同遗传背景NK细胞对转化细胞杀伤效应的差异。方法:实验于2007-03/09在南方医科大学珠江医院血液科完成。①材料:SPF级8～10周龄的C57BL/6(H-2b)小鼠10只、Balb/C(H-2d)小鼠10只、Balb/C(H-2d)×C57BL/6(H-2b)杂交F1代(H-2d/b)小鼠10只,均购于南方医科大学实验动物中心,实验过程中对动物的处置符合动物伦理学标准。YAC-1细胞株由本室冻存;C57BL/6鼠源性红白血病细胞株FBL-3由周健博士惠赠。②实验方法:脱臼处死3种不同遗传背景小鼠,无菌取脾,密度梯度离心法常规分离脾单个核细胞,免疫磁珠细胞阳性分选得到3种不同遗传背景小鼠的NK细胞,即全相合C57BL/6组、不相合Balb/C组、半相合F1代组,以其作为效应细胞。将培养至对数生长期的YAC-1、FBL-3细胞株以及ConA刺激的C57BL/6小鼠脾细胞作为靶细胞。相对于靶细胞,不相合Balb/C组与半相合F1代组为同种异体反应性NK细胞,全相合C57BL/6组为自体NK细胞。③实验评估:通过流式细胞术检测CD49b+的表达鉴定NK细胞纯度。调整靶细胞YAC-1密度为2×108L-1,按效靶比5:1,10:1,20:1,40:1以乳酸脱氢酶释放法测定NK细胞的杀伤活性。调整靶细胞FBL-3和ConA刺激的C57BL/6小鼠脾细胞密度均为2×108L-1,按效靶比20:1,40:1同法测定NK细胞的杀伤活性。结果:①NK细胞的纯度鉴定:CD49b+细胞率全相合C57BL/6组为(90.93±2.46)%,不相合Balb/C组为(89.50±1.04)%,半相合F1代组为(91.93±2.01)%,分选所得NK细胞的纯度满足实验要求。②NK细胞对YAC-1细胞的杀伤活性:3组NK细胞的杀伤活性均随效靶比5:1,10:1,20:1,40:1的升高而增强;同一效靶比时3组NK细胞对YAC-1的杀伤活性差异无显著性意义(F=0.557,P=0.600;F=0.550,P=0.604;F=0.503,P=0.628;F=0.946,P=0.439)。③NK细胞对FBL-3和ConA刺激的C57BL/6小鼠脾细胞的杀伤�AIM： Cell mediated immunotherapy focuses not only on killing abnormally growing tumor cells, but also on dealing with potent transformed cells. We observe the differences between cytotoxic effects against transformed cells exerted by natural killer （NK） cells from different genetic backgrounds. METHODS： Experiments were performed at Department of Hematology, Zhujiang Hospital, Southern Medical University from March to September 2007. C57BL/6（H-2^b）, Balb/C（H-2^d）, Balb/C（H-2^d）×C57BL/6（H-2^b） hybrid F1（H-2d^/b） mice were selected in the study. The sample number for each strain was 10, SPF, 8-10 weeks, obtaining from experimental animal center in Southern Medical University. Treatment for the experimental animal met ethical standards. YAC-1 cell line was preserved at our lab; the FBL-3 （erythroleukemia cell line derived from C57BL/6 mouse strain） was endowed by Ph.D. Zhou. Three different strains of mouse were sacrificed by luxation, then its spleen was got sterilely. Splenocytes were obtained by density gradient centrifugation as usual. NK cells from different genetic backgrounds were harvested by MACS positive sorting as effector cells. That is, all-identical C57BL/6, non-identical BALB/c and haplo-identical F1. Take YAC-1, FBL-3 and ConA blasted splenocytes as target cells. So the non-identical BALB/c and haplo-identical F1 were allo-reactive NK cells, while the all-identical C57BL/6 was self NK cells. The purifications of isolated NK cells （CD49b^＋ expression） were detected by flow cytometry （FCM）. Take the YAC-1, FBL-3 and ConA blasted splenocytes at the density of 2×10^8 L^-1 as target cells. Cytotoxic effects exerted by NK cells were detected by lactate dehydrogenase （LDH） releasing assay at the E/T ratio of 5：1, 10：1, 20：1, 40：1 （as for YAC-1） and 20：1, 40：1 as for the FBL-3 and ConA stimulated splenocytes. RESULTS： The purifications of isolated NK cells： The ratio of CD49b^＋ cells from the C57BL/6 group was （90.93±2.46）%, from the

关 键 词：自然杀伤细胞 遗传背景 细胞免疫治疗 

分 类 号：R617[医药卫生—外科学]