IL-2／IL-15刺激后脐血NK细胞穿孔蛋白表达及其与细胞毒活性的关系  

Expression of Perforin in Cord Blood NK Cells after IL-2/IL-15 Stimulation and Its Relation with Cytotoxicity

作　　者：吴燕峰[1] 张碧红[2] 岑丹阳[2] 魏菁[3] 陈纯[1] 

机构地区：[1]中山大学孙逸仙纪念医院生物治疗中心,广东广州510120 [2]中山大学孙逸仙纪念医院儿科,广东广州510120 [3]中山大学孙逸仙纪念医院医学研究中心,广东广州510120

出　　处：《中国实验血液学杂志》2011年第4期1015-1018,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号30772367);广东省自然科学基金(编号8151008901000099);广东省科技计划资助项目(编号2008B0303012066)

摘　　要：本研究旨在探讨脐血NK细胞的穿孔蛋白表达水平以及IL-2、IL-15作用下穿孔蛋白表达与NK细胞细胞毒活性的关系。采用CD3阴选后CD56阳选两步免疫磁珠分选法纯化脐血NK细胞,流式细胞术测定NK细胞纯度,通过IL-2、IL-2+IL-15两种细胞因子培养体系培养3天,流式细胞术测定培养前后穿孔蛋白表达水平;LDH释放法检测培养前后脐血NK细胞对白血病细胞株K562的细胞毒活性(效靶比10∶1)。结果表明,纯化后CD3-CD56+细胞含量为(92.39±0.8)%。,以K562为靶细胞,新鲜纯化CB-NK的杀伤活性为(27.76±8.8)%,IL-2组杀伤活性为(61.9±9.1)%,IL-2+IL-15组杀伤活性为(87.62±3.7)%,新鲜纯化CB-NK穿孔蛋白表达率为(67.21±6.14)%,IL-2组CB-NK细胞穿孔蛋白表达率(84.55±3.8)%,IL2-+IL-15组CB-NK细胞穿孔蛋白表达率(87.22±2.2)%。穿孔蛋白表达率与NK细胞毒活性呈正相关(r=88.6,p<0.05)。IL-2组穿孔蛋白表达率与新鲜组差别有统计学意义,IL-2组与IL-2+IL-15组穿孔蛋白表达率差别无统计学意义。结论:促进穿孔蛋白表达是IL-2提高脐血NK细胞细胞毒活性的途径之一。This study was aimed to investigate the expression level of perforin in cord blood NK cells and the relation of perforin expression after IL-2, IL-15 stimulation to cytotoxicity of NK cells. NK cells were isolated from cord blood MNC by depleting CD3 ＋ cells and then enriching CD56 ＋ cells using immunomagnetic separation （ CD3 and CD56 cell isolation kit, autoMACS, miltenyi）. The purity was analysed by flow cytometry. According to the different combination of cytokines, there were two groups： group A （IL-2） and group B （IL-2 ＋ IL-15 ）. The cytotoxicity and perforin expression rate of fresh and different cultured CB-NK cells against K562/Jurkat cell lines were estimated by LDH release test（cytotox 96 non-radioactive cytotoxicity assay）. The results showed that the purity of NK cells after separation was more than 90%. The cytotoxicity towards both tumor lines in group B was higher than that in group A （p 〈0.05 ）, and cytotoxicity in group A was higher than that of fresh NK cells （p 〈 0. 05 ）. Perforin expression rate of group A （84.55%） was higher than that of fresh NK cells （67.21%） （p 〈 0.05）, and there was no significant difference between group A and B （84.55% versus 87.22% ） Cytotoxic activity of CB-NK cells was positively correlated with perforin expression rate （ r = 0.886, p 〈 0.05 ）. It is concluded that IL-2 can enhance cytotoxicity of CB/BM-NK cells by increasing the perforin expression.

关 键 词：脐血 IL-2 IL-15 自然杀伤细胞 穿孔蛋白 细胞毒 

分 类 号：R730.3[医药卫生—肿瘤] R730.51[医药卫生—临床医学]