云芝多糖增强人NK细胞杀伤功能的体外研究  

Enhance effect of polysaccharide K(PSK) on killing activity of human nature killing cells in vitro

作　　者：孙飞[1] 陶正中 周忠海[3] 吕小婷[3] 陈娜云[3] 陈玲[3] 姚仁南[1] 

机构地区：[1]中国人民解放军第九七医院输血科,江苏徐州221004 [2]中国人民解放军第九七医院临床检验科,江苏徐州221004 [3]中国人民解放军第九七医院实验中心,江苏徐州221004

出　　处：《生物医学工程与临床》2017年第2期126-131,共6页Biomedical Engineering and Clinical Medicine

基　　金：南京军区医学科技创新重点课题(14ZD17)

摘　　要：目的探讨云芝多糖(PSK)对体外培养的人自然杀伤(NK)细胞杀伤功能的影响。方法采集健康成年人外周血,分离单个核细胞(PBMC),加入含白细胞介素(IL)-2的NK细胞培养液。培养第10天时加入不同质量浓度(100、75、50、25、10、5μg/m L)的PSK诱导NK细胞48 h,收集细胞检测。用CCK-8法检测细胞增殖情况,流式细胞仪检测诱导前后NK细胞穿孔素、颗粒酶B、CD107a及NKG2D受体表达(PSK诱导为实验组,同时设不加药物及无细胞的空白对照孔为对照组)。通过CCK-8法检测对红白血病细胞株K562细胞的杀伤活性:取质量浓度25μg/m L PSK诱导48 h后NK细胞,调整细胞浓度至1×10~9/L作为效应细胞;调整红白血病肿瘤细胞株K562细胞浓度至5×10~7/L,按效靶比为5∶1、10∶1、20∶1、40∶1、80∶1的比例混合培养于96孔板内,同时设单独效应细胞孔、单独靶细胞孔(实验组)和空白孔(对照组)。结果培养10 d后NK细胞纯度达到78.70%左右。经PSK诱导48 h后,NK细胞的增殖及功能具有一定浓度依赖性,PSK质量浓度在25μg/m L对NK细胞的增殖率(51.62%±3.20%)最为明显(P<0.01),之后逐渐下降。在质量浓度0~100μg/m L可以促进NK细胞的生长和增加NK细胞表面穿孔素、颗粒酶B、CD107a和NKG2D受体表达,以及增强对红白血病细胞株K562细胞的杀伤活性;尤其是当PSK质量浓度为25μg/m L时,颗粒酶B、穿孔素、NKG2D和CD107a表达为55.42%±2.34%、70.49%±1.87%、83.45%±1.77%和85.00%±2.02%,显著高于对照组(25.83%±1.31%、40.79%±1.59%、70.66%±2.39%和72.79%±2.31%)(P<0.01)。在效靶比80∶1时,实验组杀伤活性为57.63%±3.42%,高于对照组(24.78%±2.47%)(P<0.01)。结论 PSK在一定质量浓度下能促进NK细胞的生长,且增强NK细胞的杀伤功能。Objective To explore the effect of polysaccharide K（PSK） on killing activity of human nature killing（NK） cells in vitro. Methods Peripheral blood mononuclear cell（PBMC） were collected and separated from healthy donors, and generated in NK cells culture medium contained interleukin-2（IL-2） for 10 days, then the purity of NK cells was detected by flow cytometer.The cells were co-cultured with different concentrations（100, 75, 50, 25, 10, 5 μg/m L） of PSK for 48-hour, then collected to be detected. The proliferation of NK cells were determined by CCK-8 assay. Flow cytometry was used to detect the perforin,granzyme B, CD107 a, NKG2 D expression of NK cells（PSK induction as experimental group, and without drug and cell-free control as control group）, and CCK-8 assay was used to measure cytotoxic activity of NK cells against cancer K562 cells. The PSK with concentration of 25 μg/m L was used to induce NK cells for 48-hour, and cell concentration was adjusted to 1 × 10^9/L as effector cells; The cell concentration of K562 cells was adjusted to 5 × 10^7/L and mixed in 96-well plate by effective target ratio of 5 ∶ 1, 10 ∶ 1, 20 ∶ 1, 40 ∶ 1, and 80 ∶ 1. The cells were devided into single-effect wells, single target wells（experimental group） and blank wells（control group）. Results The purity of NK cells from different individual cultured for 10 days reached approximately 78.70 %. After treated with various concentrations of PSK after 48-hour, the proliferation and function of NK cells were depended on the concentration, the proliferation rate of NK cells at 25 μg/m L of PSK concentration reach the highest（51.62 % ± 3.20 %）（P〈0.01） and then gradually decreased. The concentration of 0-100 μg/m L, PSK was promoted NK cells growth and increased the perforin, granzyme B, CD107 a, NKG2 D expression of NK cells, and enhanced the cytotoxicity of NK cells against K-562 cells. Especially, at concentration of 25 μg/m L, the expression of granzyme B, perforin,NKG2 D and CD107

关 键 词：云芝多糖 NK细胞 细胞增殖 杀伤功能 

分 类 号：R285.6[医药卫生—中药学]