Effect And Mechanism Of Interleukin 21 On Cytokine Induced Killer Against Leukemic K562 Cells

Objective:To observe effect of interleukin 21 induced Cytokine induced killer cells' proliferation,secretion,the proportion of cells subsets,the m RNA expression of Cytokine factors and explore impact of interleukin 21 on ant-Leukemic K562 cells activity of Cytokine induced killer cells and the mechanism.Methods:Collected 30 ml heparin anticoagulated peripheral blood from healthy volunteers.Peripheral blood mononuclear cells were extracted by Ficoll solution.The cells were placed in three different sterile flasks and randomly divided it into control group(IL-2 100 ng/ml,no IL-21),IL-21 group(no IL-2,IL-21 100 ng/ml)and IL-21+IL-2 group(IL-2 100 ng/ml,IL-21100 ng/ml).They were cultured by 10% FBS1640 medium.The morphological changes were observed by inverted phase contrast microscopy every day.The number of cells was counted by manual counting at 0th,7th,10 th and 14 th days.At 14 th day,the supernatant were collected and test IFN-?,TNF-?,IL-2,IL-10,TGF-?,IL-21 by Elisa assays,the proportion of CD3+CD4+,CD3+CD8+,CD3+CD56+,CD25+and CD4+CD25+ cells subsets by Flow cytometry and the m RNA expression of perforin,granzyme A,granzyme B,Fasl,NKG2 D,IL-2R and IL-21 R were examined by RT-PCR for all group of CIK cells.At the same time,the toxicity of Leukemic K562 cells was measured by CCK-8.JAK-STAT signaling pathway of CIK cells were measured by Western blot on the same day.Rusults:When CIK cells were cultured on the 5th day,the cell volume increased slightly and the number increased gradually.On 7th and 10 th day,the number of cellswas remarkable increased,was significantly higher IL-21 group and IL-21+IL-2 group than control group(P<0.05).On the 14 th day,the number of IL-21 group was significantly lower than that of control group(P<0.05).There was no significant difference between the IL-21+IL-2 group and the control group(P>0.05).In 14 th day,the secretion of TNF-?,IFN-?,IL-21 both IL-21 group and IL-21+IL-2 group is higher significant than control group(P<0.05),IL-10 is lower significant than control group(P<0.05),IL-2 and TGF-? have no significant differences(P>0.05).The proportion of CD25+,CD3+CD4+ and CD3+CD56+ cells subsets is higher significant than control group(P<0.05),the proportion of CD3+CD8+ and CD4+CD25+ cells subsets have no significant differences(P>0.05),the cytotoxity of CIK cell on K562 is higher significant than control(P<0.05).The m RNA expression of perforin,granzyme B,Fasl,IL-2R and IL-21 R is higher significant than control group(P<0.05).The m RNA expression of granzyme A,NKG2 D have no significant differences(P>0.05).STAT3,STAT5 b signaling pathway of IL-21-CIK cells is higher significant than control.STAT1,STAT5 a signaling pathway of IL-21-CIK cells have no significant differences.Conclusion:IL-21 induced CIK cells without promoting proliferation,but increasing the percentage of CD25+,CD3+CD4+,CD3+CD56+subgroups,promoting secretion of IFN-?,TNF-?and IL-21,inhibiting IL-10 secretion and Promoting the expression of perforin,granzyme B,Fasl,IL-2R and IL-21 R m RNA.Thereby it can promots leukemia K562 cells toxicity of IL-21 induced CIK cell.It is one of the mechanisms that JAK-STAT STAT3,STAT5 b signaling pathway is activated by IL-21.