IL-23诱导人外周血单个核细胞增殖及其体外杀伤MUTZ-1细胞的实验研究

目的：观察IL-23诱导人外周血单个核细胞（ PBMNC）增殖及其对骨髓增生异常综合征（ MDS）细胞系MUTZ -1的杀瘤作用。方法采用密度梯度离心法分离获得正常人PBMNC，细胞分4组：阴性对照组（未加IL-23），3个浓度IL-23组（2、10、50 ng／ml），体外诱导72 h，并与MUTZ-1共培养。流式细胞术检测不同时间诱导后PBMNC细胞增殖和表型变化。乳酸脱氢酶（ LDH）释放法检测PBMNC对MUTZ-1细胞的杀瘤效果。 Real time-PCR和Western-blot检测PBMNC细胞培养上清液中IFN-γ、TNF-α、TGF-β、颗粒酶A、颗粒酶B、穿孔素mRNA和蛋白表达。结果体外诱导培养1、3、5、7 d时，IL-23诱导能够明显促进PBMNC细胞增殖，呈时间和浓度依赖性，与阴性对照组比较差异有统计学意义（ P ＜0．05）；CD3＋、CD4＋、CD5＋6阳性细胞数明显增加，CD8＋阳性细胞数明显降低，且IL-23作用后的PBMNC均对MUTZ-1细胞产生良好的杀瘤活性；与阴性对照组比较，IL-23作用后PBMNC细胞上清液中IFN-γ、TNF-α、TGF-β、颗粒酶A、颗粒酶B、穿孔素mRNA和蛋白表达明显增加，呈时间和浓度依赖性，差异有统计学意义（ P ＜0．05）。结论 IL-23能够明显促进PBMNC细胞增殖，并通过诱导PBMNC细胞表达IFN-γ、TNF-α、TGF-β、颗粒酶 A、颗粒酶B、穿孔素发挥杀伤MUTZ-1细胞作用。

Experimental study on the effects of IL-23 in inducing proliferat ion of human peripheral blood mononuclear cell and in killing MUTZ-1 cells in vitro

Objective To observe the effects of IL -23 in inducing proliferation of human peripheral blood mononuclear cell ( PBMNC) and in killing MUTZ-1 cells—a human myelodysplastic syndrome ( MDS) cell line in vitro. Methods The PBMNCs were separated by ficoll density gradient centrifugation.The experiment was divided into control group (without IL-23), IL-23 high,median,low dose groups (2, 10, 50ngm/l,respectively) in which the PBMNCs were induced by IL-23 for 72 hours in vitro.Then PBMNCs were co-cultured with MUTZ-1 cells.The proliferative ability of PBMNC cells was detected by flow cytometry, and the killing effect of PBMNC on MUTZ-1 cells was measured by LDH releasing method . Moreover the expression levels of IFN-γ, TNF-α, TGF-β, granzyme A, granzyme B and perforin at mRNA and protein levels were detected by Real time-PCR and Western Blot,respectively.Result s After 1, 3, 5,7 -day induction, IL-23 could obviously promote the proliferation of PBMNCs,with a concentration and time-dependent manner, as compared with that in control group ( P <0.05).The positive cell numbers of CD3+, CD4+and CD5+6 were significantly increased, however, CD8+cells were obviously decreased,moreover,the killing effects of PBMNC induced by IL-23 on MUTZ-1 cells were increased. After treated by IL-23, the expression levels of IFN-γ, TNF-α, TGF-β, granzyme A, granzyme B and perforin at mRNA and protein levels were significantly increased , with a concentration and time -dependent manner, as compared with those in control group ( P <0.05).Conclusion IL -23 can obviously promote the proliferation of PBMNCs and can kill MUTZ-1 cells through inducing the expressions of IFN-γ, TNF-α, TGF-β, granzyme A, granzyme B and perforin of PBMNCs.