NK细胞体外培养及对HepG2细胞的杀伤活性研究

Study of the Effect of NK Cells Cultured in Vitro and Its Killing on HepG2 Cells

目的:研究NK细胞的体外培养及对HepG2的杀伤活性。方法:采集外周血,肝素抗凝,分离单个核细胞(PMBC),用NK细胞培养基(补加IL-2200U/ml)培养12d,用流式细胞仪检测其表面标志及细胞毒颗粒表达水平,并按效靶比5∶1、10∶1、20∶1加入HepG2细胞,比较其杀伤活性。结果:培养12d的NK细胞CD3-CD56+阳性率(73.6±11.3)%与培养前的阳性率(14.2±5.1)%相比有显著性差异(P<0.001),其穿孔素、颗粒酶B、CD107a的表达水平分别为(75.5±10.9)%、(62.2±8.9)%、(63.9±10.5)%。在5∶1~20∶1效靶比范围内,随着效靶比增加,NK对HEPG2细胞杀伤活性增强(P<0.05)。结论:NK细胞对肿瘤细胞具有杀伤活性,随着效靶比增加,杀伤活性增强。

Objective:To study of the cytotoxicity effect of natural killer(NK)cells on HepG2 in vitro.Methods:Peripheral blood was collected for heparin anticoagulated,mononuclear cells(PMBC)were isolated,cultured with NK cell culture medium(supplemented with IL-2200 U/ml)for 12 days.Flow cytometry was used to detect the expression level of surface markers and cytotoxic granules.HepG2 cells were added at a ratio of 5∶1,10∶1 and 20∶1 to compare their killing activity.Results:The positive rate of CD3-CD56+in NK cells cultured on 12d(73.6±11.3)%was significantly different from that before culture(14.2±5.1)%(P<0.001),the expression levels of perforin,granulase B and CD107a were(75.5±10.9)%,(62.2±8.9)%and(63.9±10.5)%respectively.In the range of 5∶1 to 20∶1 target ratio,the cytotoxicity activity of NK on HepG2 cells was enhanced with the increase of the effective target ratio(P<0.05).Conclusion:NK cells have cytotoxic activity against tumor cells,and the cytotoxicity increases with the increase of the effective target ratio.