内源性表达白细胞介素21的CIK细胞的抗白血病作用及其机制

Effects and mechanism on anti-leukemic activity of cytokine-induced killer cells with an endogenousexpression of interleukin-21

目的研究内源表达白细胞介素2l（IL-21）对外周血来源的细胞因子诱导的杀伤细胞（CIK细胞）抗自血病的作用及其作用机制。方法采集分离健康人外周血单个核细胞，加用细胞因子诱导培养CIK细胞，构建表达IL-21基因的慢病毒载体，感染CIK细胞并鉴定后，四甲基偶氮唑盐（Mqq＂）法检测CIK细胞的增殖能力及杀伤白血病细胞系K562细胞作用；半定量反转录（RT）-PCR法检测CIK细胞干扰素1（IFN-y）、肿瘤坏死因子0（TNF．OL）、TNF．B、穿孔素、颗粒酶A、颗粒酶B、Fas配体（FasL）和NKG2D分子（NKG2D）的mRNA表达；流式直接免疫标记法检测CIK细胞免疫表型、细胞表面IL-21受体（IL．21R）、FasL及细胞内穿孔素、颗粒酶B的表达；酶联免疫法检测培养上清中IFN-^v和TNF-OL的表达。结果经酶切与测序鉴定，IL-21慢病毒载体构建正确，共转染CIK细胞中有目的基因IL-21的表达。（1）与空载体组相比，内源表达IL-21组总的细胞增殖之间差异无统计学意义（P=0．856），内源表达IL-21组CIK细胞阳性细胞数高于空载体组（24．60％±2．10％比16．95％±4．70％，P=0．042）。（2）内源表达IL．21组CIK细胞对K562细胞的杀伤率高于空载体组（58．4％±8．3％比23．3％±2．8％，P=0．009），作用第5天时，杀伤作用仍能维持在61．2％±6．2％（P：0．003），相比外源性IL-21作用的CIK细胞的杀伤率（44．6％±8．3％比22．8％±2．8％，P=0．034）更强，而且作用时间更长久。（3）内源表达IL-21组CIK细胞表面IL-21R的表达比空载体组增加约2倍。（4）与空载体组比较，内源表达IL-21组CIK细胞IFN--,／和TNF-仪mRNA的表达升高约1．5倍，穿孔素、颗粒酶B、FasLmRNA的表达量升高近2倍，颗粒酶A、TNF-B和NKG2DmRNA的表达与空载体组差异无统计学意义。（5）对mRNA表达差异有统计学意义的指标进-步行蛋白质表达的检测发现：内源表达IL-21组CIK细胞表面FasL、穿孔素、颗粒酶B、IFN-y、TNF-a的表达水平均高于空载体组0．56％±0．37％比0．06％±0．02％、25．86％±6．13％比12．23％-4-2．35％、37．58％±2．30％比14．56％±1．36％、（55．3±3．5）ng／L比（23．2±5．6）．g／L、（15．6±0．6）斗∥L比（5．6±0．6）μg／L，均P〈0．05]。结论内源表达IL，21的CIK细胞具有更强的抗白血病作用，而且作用时间更长久，此作用可能是通过增加其受体的表达，增加穿孔素、颗粒酶B、FasL、IFN-y和TNF-a.

Objective To explore the effects and mechanism on anti-leukemic activity of cytokine inducing killer （CIK） cells with an endogenous expression of interleukin-21 （IL-21）. Methods Mononuclear cells were isolated from peripheral blood and cultured with cytokines to generate CIK cells.IL-21 lentiviral vector was constructed and used to transfect 293T cells. Then the culture supernatant with virus infected CIK cells was identified. Proliferation of CIK cells and their cytotoxic activity against K562 cells were measured by methyl thiazolyl tetrazolium （ MTT ）. The expressions of interferon-y （ IFN-y）, tumor necrosis factor-a（TNF-ca） , tumor necrosis factor-β（TNF-β） , perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Immunophenotypes of CIK cells, IL-21 receptor （IL-21R） and FasL on the surface of CIK cells, intra-cellular perforin and granzyme B of CIK cells were measured by flow eytometry. And the concentrations of IFN-a/and TNF-ot in cultured supernatant were measured by enzyme immunoassay. Results By restriction enzyme digestion and sequencing, IL-21 lentiviral vector was identified, after transfecting virus surpernatant into CIK cells, the expression of IL-21 was detected in CIK cells. Compared to control, （1） the total number of cells remained unchanged, but the proportion of cells expressing CD3 ＋/CD56＋ phenotype increased from 16. 95%±4. 70% to 24. 60%± 2. 10% . （2） Cytotoxic activity against K562 cells by CIK cells increased from 23.3%±2. 8% to 58. 4%±8.3% and stayed at 61.2%±6. 2% after 5 days. It was stronger and longer compared to the exogenous effect of IL-21 （ from 22. 8%±2. 8% to 44. 6%±8.3% ）. （3） The expression of IL-21R increased around 2 folds. （4） The mRNA expressions of IFN-y, and TNF-ct increased almost I. 5 folds, perforin, granzyme B, FasL rose almost 2 folds, the expressions of granzyme A, TNF-~ and NKG2D were similar with those of controls. （ 5 ） Detected by flow cytometry, the expression of FasL of CIK cells was higher than that of control （0. 56%±0. 37% vs 0. 06% ±0. 02% ） , the expression of perforin increased from 12. 23%±2. 35% to 25.86%±6. 13% , the expression of granzyme B rose from 14. 56%±1.36% to 37.58%±2. 30%, the concentration of IFNy in culture supernatant spiked from （23.2 ±5.6） to （55.3±3.5） ng/L and TNF-a jumped from （5.6±0. 6） to （15. 6 ± O. 6 ） μg/L. Conclusions CIK cells with an endogenous expression of IL-21 have stronger anti- leukemic activity through an up-regulation of IL-21 R, perforin, granzyme B, FasL, IFN-y, and TNF-y Thus IL-21 mac potentially enhance the anti-leukemic immunotheraDv.