IL2、IL12、IL15的不同组合对人外周血源NK细胞功能的影响

本研究旨在探讨细胞因子IL2，IL12，IL15的不同组合对体外扩增的人外周血来源的NK细胞功能的影响。IL2、IL12和IL15等细胞因子的不同组合培养的人外周血NK细胞分为IL2组、IL2＋IL12组、IL2＋IL15组、IL2＋IL15＋IL12组；无细胞因子培养的NK细胞组为对照组。用细胞计数盒分别测定不同效靶比下NK细胞对靶细胞（K562）的杀伤作用；用ELISA方法检测培养液上清中IFN-γ的分泌水平；用建立竞争性RT—PCR方法定量检测穿孔蛋白、颗粒酶BmRNA的表达水平。结果显示，各组培养的NK细胞对K562细胞的杀伤率增强，在不同效靶比下IL2＋IL15和IL2＋IL15-4-IL12组NK细胞对K562细胞的杀伤率显著高于其他组，但此两组间无显著性差异（P〉0．05）；各细胞因子组培养液中分泌IFN-γ水平均显著高于未加细胞因子的空白对照组（P〈0．01），在不同细胞因子条件下，IFN-γ分泌水平依次为IL2＋IL12＋IL15组〉IL2＋IL12组〉IL2＋IL15组〉IL2组（P〈0．01）；各培养组的NK细胞穿孔蛋白及颗粒酶B基因的表达量均较对照组明显增加（P〈0．01），且IL2＋IL15组和IL2＋IL12＋IL15组上述两种基因的表达量均显著高于其他组（P〈0．01），但组间差别较小，与NK细胞杀伤实验的结果相一致。结论：不同细胞因子组合诱导的外周血NK细胞功能有差异。IL2和IL15在增强NK细胞杀伤功能及促进其扩增作用方面有协同作用，IL12主要刺激NK细胞分泌细胞因子（如IFN-γ），增强其免疫调节功能。

Effect of Various Combinations of IL2, IL12 and IL15 on Function of Human Peripheral Blood Derived NK Cells

This study was purposed to explore the changes in biological functions of human peripheral blood derived NK Cells after ex vivo expansion with different combinations of interleukin IL2 and/or IL12, IL15 . According to different combination of cytokines, cultured NK cells were divided into 4 groups： group IL2, group IL2 ＋ IL12, group IL2 ＋ IL15 and group IL2 ＋ IL15 ＋ IL12. The group in which NK cells were cultured without cytoldnes was used as control. The cytotoxicity of cultured NK cells to target K562 cells was determined by using cell counting kit-8 ; the level of IFN-γ in supernatants of NK cell culture was detected by ELISA; the perforin and granzyme B mRNA expressions were assayed by competitive quantitative RT-PCR. The results showed that the cytotoxicity of expanded NK cells in groups cultured with cytokines at different E： T ratio was significantly higher than that in group without cytokines （p 〈 0. 01 ）, although the cytotoxicity of NK cells in IL2 ＋ IL15 ＋ IL12 group seem to be slightly higher than that in IL2 ＋ IL15 group, but there was no statistic difference （p 〉 0.05 ）. The IFN-γ levels in the supernatants of NK cell culture in the presence of cytokines significantly increased, and the IFN-γ levels in IL2 ＋ IL15 ＋ IL12 group and IL2 ＋ IL12 group were significantly higher than that in others（p 〈0.01 ）. The expressions of perforin and granzyme B mRNA of expanded NK cells in groups cultured with cytokines was significantly higher than that in control group （p 〈0.01 ）, and was consistant with cytotoxicity of NK cells. It is concluded that there are differences in the functions of NK cells cultured with different cytokines. IL2 and IL15 have synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion. However, the main function of IL12 promotes NK cells to secrete IFN-γ, which plays a role in immunoregulation.