猪苓多糖联合白细胞介素15增强人外周血单个核细胞对K562细胞杀伤活性的影响

Study of PPS combined with interleukin-15 enhanced killing activity of peripheral blood mononuclear cells to K562 cells

目的研究猪苓多糖联合白细胞介素(interleukin,IL)15对人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)增殖的影响,探讨该效应细胞对人慢性髓细胞白血病K562细胞株增殖的抑制作用及可能的机制。方法密度梯度法分离PBMC,分别应用IL-2、IL-15、猪苓多糖、猪苓多糖+IL-2及猪苓多糖+IL-15刺激PBMC增殖,获得效应细胞,流式细胞仪(FCM)检测PBMC中T细胞、NK细胞活化的免疫表型;双抗体夹心酶联免疫吸附测定(ELISA)法检测效应细胞培养上清中干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)的水平;四甲基偶氮唑蓝(MTT)比色法检测效应细胞对靶细胞的细胞毒作用。结果猪苓多糖联合IL-15能刺激PBMC中T细胞及NK细胞增殖,在促进NK细胞增殖作用上具有协同作用;能上调效应细胞培养上清中IFN-γ和TNF-α的水平,且具有协同作用;并能增强效应细胞对靶细胞的杀伤活性,大致呈时间-效应趋势。结论猪苓多糖+IL-15能够增强PBMC对K562细胞株的细胞毒作用,表现为肿瘤细胞增殖抑制率(IR)提高,杀伤机制可能与其刺激T细胞及NK细胞增殖,同时细胞培养上清中细胞因子IFN-γ和TNF-α的水平提高有关。

Objective To study the effects of polyporus polysaccharide（PPS）combined with interleukin-15（IL-15）on the proliferation of human peripheral blood mononuclear cells（PBMC）,and to further clarify the effect on its killing activity and mechanism to chronic myeloid leukemia K562 cells.Methods Density gradient method was used to separate PBMC,then IL-2,IL-15,PPS,PPS combined with IL-2and PPS combined with IL-15 were used to stimulate PBMC to proliferate PBMC respectively,so as to acquire the effecter cells.Finally,FCM was used to detect the immunophenotype of T cells,NK cells activated in PBMC;double antibody sandwich ELISA method was performed to detect the levels of IFN-γand TNF-αin cell culture supernatant; MTT colorimetry method was performed to detect the cytotoxic effect of effector cells to target cells.Results The combination of PPS and IL-15 could synergistically stimulate T cells and NK cells＇ proliferation in PBMC;it could raise the levels of cytokines IFN-γand TNF-αin effector cell culture supernatant;Also it could enhance the cytotoxic effect of effector cells to target cells in a time-dependent manner.Conclusion The combination of PPS and IL-15 can improve the cytotoxicity effect of PBMC on cell strain K562 which presented at the increase of IR of the tumor cells＇ proliferation.The killing mechanism may be caused by the proliferation of T cells and NK cells stimulated by the combination of PPS and IL-15 and the improved levels of IFN-γand TNF-αin cell culture supernatant.