PHA诱导的CIK细胞的生物学特性及其对K562细胞杀伤活性影响的研究

Biological Characteristics of PHA-induced CIK Cells and Its Killing Activity to K562 Cells

本研究探讨植物血凝素(PHA)诱导的细胞因子诱导的杀伤细胞(CIK)与传统方法制备的CIK细胞的体外增殖能力、效应细胞含量和对K562细胞杀伤活性影响并分析其差异。分离健康人外周血单个核细胞(PBMNC),分甲、乙两组,其中甲组用传统培养方法从PBMNC制备的传统CIK细胞,乙组采用PHA诱导单个核细胞制备获得的新型CIK细胞。在培养过程中,每3 d统计各组细胞培养体系中细胞活率和细胞绝对值,并在培养至第15天时,用流式细胞仪分别检测两组细胞免疫表型,统计CD3+CD56+、CD3+CD8+和CD3+CD4+细胞占各培养体系总细胞数的比例;同时用CCK-8试剂盒分别检测两组细胞在不同效靶比时对K562细胞的杀伤活性。结果表明:采用PHA诱导制备新型CIK细胞的方法比传统方法更能促进细胞增殖(P<0.05),且细胞活率都保持在90%以上。两组CD3+CD8+、CD3+CD56+细胞比例都显著升高。与传统方法比,新方法的CD3+CD8+细胞升高比例存在显著差异(P<0.05),CD3+CD56+细胞提升比例不存在差异,同时CD3+CD4+下降的比例也不存在差异。效靶比为5∶1、10∶1、20∶1、40∶1时,PHA诱导制备的新型CIK细胞比传统方法制备的CIK细胞对K562细胞的杀伤活性更强(P<0.05),且随着效靶比的升高两种效应细胞对K562细胞的杀伤活力的差异明显性也增加。结论:与传统方法相比,PHA可明显提高CIK细胞的增殖能力,调高CD3+CD8+细胞的比例,增强CIK细胞对K562细胞的杀伤活性,这为白血病及其他肿瘤的细胞免疫治疗提供一种新来源的CIK细胞和可靠依据。

The purpose of study was to investigate the in vitro proliferation ability of PHA-induced CIK ceils and traditionally prepared CIK cells,the effector cell level and its influence on killing activity to K562 cells,and to analyze the difference between them.The peripheral blood mononuclear cells （PBMNC） of healthy persons were isolated and divided into A and B group.The CIK cells in A group were obtained by using traditional culture method,the CIK cells in B group were prepared by PHA induction.During the cultivation,the cell survival rate and cell absolute value in the cell culture system were counted every 3 days.On day 15 of culture,the cell immunophemotype of 2 groups were detected by flow cytometry,and the ratios of CD3 ＋ CD56 ＋,CD3 ＋ CD8 ＋ and CD3 ＋ CD4 ＋ cells in total cell amount of culture system wre accounted.Meantime,the killing activity to K562 cells in different effector-target ratios was detected by using CCK-8 kit between the 2 groups.The results showed that the method of preparing CIK by PHA induction promoted the cell proliferation nore than that of the traditional method （P ＜ 0.05）,moreover,both the survival rate of cells in 2 groups was more than 90％.The CD3 ＋ CD8 ＋,CD3 ＋ CD56 ＋ cell ratio in 2 groups obviously increased.As compared with traditional method,the CD3 ＋ CD8 ＋ cell level in B group was enhanced （P ＜ 0.05） ; but there were no statistical differences in increase of CD3 ＋ CD56 ＋ cell level and decrease of CD3 ＋ CD4 ＋ cell level between 2 groups.while the effector-target ratio is 5∶ 1,10∶ 1,20∶ 1 and 40∶ 1,the killing activity of PHA-induced CIK cells to K562 cells was more stronger than traditionally-prepared CIK cells （P ＜ 0.05）,moreover,along with increase of effector-target ratio,the difference of killing activity to K562 cells in 2 groups significantly increased.It is concluded that compared with traditional method for preparing CIK cells,the new way by PHA induction can increase the proliferation of CIK cells obviously,enhance the ratio of CD3 ＋ CD8 ＋ cells and strengthen the killing activity to the K562 cells.This new way provides a new source of CIK cells and retiable evidence for cytoimmune therapy of leukemia and other tumors.