NK细胞联合当归多糖对结肠癌细胞的体外协同杀伤作用及机制研究

Synergistic killing effect of natural killer cells combined with Angelica sinensis polysaccharide against colon cancer cells in vitro and its mechanism

目的探讨NK细胞联合当归多糖(ASP)对结肠癌细胞的体外协同杀伤作用及机制。方法分离人脐带血单个核细胞,体外采用灭活K562细胞联合细胞因子扩增获得高纯度NK细胞,设置control组、不同浓度ASP组、NK Cell组、NK Cell+ASP组、NK+SW480组、ASP+SW480组;MTT法检测不同质量浓度ASP对结肠癌细胞、NK细胞增殖的影响,酶联免疫吸附法(ELISA)检测NK细胞培养上清液中TNF-α、IFN-γ水平,钙黄素-AM释放法检测NK细胞联合ASP对结肠癌细胞的杀伤作用,流式细胞仪检测MICA、MICB、uLBP1表达率。结果与control组比较,各浓度ASP组在24 h、48 h、72 h的NK细胞数均显著升高(P<0.05),ASP质量浓度为200.00 mg/ml时,NK细胞数目最高;与control组比较,各浓度ASP组在24 h、48 h、72 h对结肠癌细胞的抑制率呈剂量性增加(P<0.05);与NK Cell组比较,NK Cell+ASP组TNF-α、IFN-γ的水平显著提高(P<0.05);ASP和NK细胞对结肠癌细胞均有杀伤作用,与单独的NK细胞和单独ASP相比,NK细胞联合ASP对SW480细胞具有更强的杀伤作用(P<0.05);与SW480组比较,ASP+SW480组SW480细胞中MICA、MICB、uLBP1的表达率显著提高(P<0.05)。结论NK细胞联合ASP体外协同杀伤结肠癌细胞,其协同作用可能与ASP促进NK细胞分泌TNF-α、IFN-γ增强其杀伤能力,促进肿瘤细胞高表达MICA、MICB、uLBP1相关。

This study was performed to investigate the synergistic killing effect and mechanism of NK cells combined with Angelica sinensis polysaccharide(ASP)on colon cancer cells in vitro.Human umbilical cord blood mononuclear cells were isolated,and high purity NK cells were obtained by inactivating K562 cells combined with cytokines in vitro.The proliferation of colon cancer cells and NK cells was detected by MTT;the levels of TNF-αand IFN-γin NK cell culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).The killing effect of NK cells combined with ASP on colon cancer cells was detected by calcein-AM release assay;while the expression rates of MICA,MICB and uLBP1 were detected by flow cytometry.Data showed that the number of NK cells in the ASP group at 24 h,48 h,and 72 h was significantly increased,as compared with control group.The concentration of NK cells was the highest when the ASP concentration was 200.00 mg/ml.The inhibition rate of colon cancer cells at 24 h,48 h,and 72 h in each concentration of ASP was increased in a dose dependent manner.The levels of TNF-αand IFN-γin ASP-treated NK cell culture medium were significantly increased.Both ASP and NK cells had a killing effect on colon cancer cells.Compared with NK cells alone and ASP alone,NK cells combined with ASP had a stronger killing effect on SW480 cells.The expression rates of MICA,MICB and uLBP1 in ASP-treated SW480 cells were significantly increased.In conclusion,NK cells combined with ASP can synergistically kill colon cancer cells in vitro,and their synergistic effects may be related to ASP promoting effects on the secretion of TNF-αand IFN-γfrom NK cells,and on the expression of MICA,MICB and uLBP1 in tumor cells.