摘要
目的采集健康志愿者和肿瘤患者的外周血单个核细胞(PBMCs)进行自然杀伤细胞(NK)的扩增培养,培养过程中白细胞介素IL-2、IL-12、IL-15、IL-18以不同组合及不同方式添加,探究体外培养NK细胞的细胞因子最佳组合和添加方式。方法根据细胞因子组合及添加方式的不同,将NK培养分为3组。A组:IL-2+IL-15组,B组:IL-2+IL-12+IL-15+IL-18组,C组:IL-12+IL-15+IL-18预处理/IL-2组。培养过程中第7天和第14天分别取样统计各组细胞因子培养后的细胞数量,并使用流式细胞仪检测NK细胞表面分子表达;将NK细胞与K562细胞共培养,然后ELISA测定NK细胞的IFNγ释放量,CSFE/7-AAD法检测NK细胞的杀伤活性。结果 3组不同的培养方式所得细胞的扩增倍数差异无统计学意义,随着培养时间的增加,NK细胞的比例也逐渐上升,3组间NK细胞比例差异无统计学意义(P>0.05)。NK细胞表面CD25(IL-2Rα)的表达情况比较,差异有统计学意义(P <0.05),C组的阳性率高于A和B组。IFN-γ的释放量和NK细胞的体外杀伤活性一致,均为C组高于A和B组。对肿瘤患者的NK细胞,C组的细胞因子添加方式也能很好的进行扩增(CD3-CD56+细胞比例大于50%)。结论 C组中的细胞因子预处理方式能够高效扩增NK细胞,与A或B组联合添加的方式比较,该方式能显著激活NK细胞的杀伤活性。
Objective To obtain the peripheral blood mononuclear cells(PBMCs) from healthy donors and tumor patients and culture natural killer(NK) cells with addition of IL-2, IL-12, IL-15 and IL-18 in various combinations and different ways so as to explore a more effective method for the in vitro expansion of NK cells. Methods Three groups of cytokine combinations and treatment patterns were established for expansion of NK cells from PBMCs, including the group A(IL-2+IL-15), the group B(IL-2+IL-12+IL-15+IL-18), and the group C(pretreatment with IL-12+IL-15+IL-18 followed by IL-2 treatment). The total cell expansion fold and proportion of NK subsets were detected on the 7 th and 14 th day. Meanwhile, the CD25 expression of NK cells was examined by flow cytometry. Then the IFN-γ production and cytotoxicity of NK cells were determined by ELISA and CFSE/7-AAD staining respectively. Results The expansion fold had no significant difference among the 3 groups. The proportion of NK cells was increasing during the expansion process, which showed a similar pattern among the 3 groups(P > 0.05). However, a progressive increase of CD25+ NK cells was observed in the groups C compared to the group A and the group B(P < 0.05). The pretreatment with IL-12, IL-15 and IL-18 for 16 h in cultures clearly stimulated more IFN-γ secretion by NK cells and enhanced NK-mediated cytotoxicity. In addition, NK cells of the tumor patients could also be efficiently expanded using pretreatment method(the proportion of NK cells was over50%).Conclusions Cytokine pretreatment method can induce highly proliferative responses and enhance NK-mediated cytotoxicity compared with the classical protocols
引文
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