CAJ | 학술논문

目的研究IL-21对外周血来源的细胞因子诱导杀伤细胞(CIK细胞)抗白血病作用及其机制.方法采集分离正常人外周血单个核细胞,加用细胞因子诱导培养,用MTT法检测CIK细胞的增殖能力及其对自血病细胞系K562细胞的杀伤作用；用流式细胞术检测CIK细胞免疫表型及其表面IL-21受体(IL-21R)、FasL、细胞内穿孔素和颗粒酶B的表达；半定量RT-PCR法检测CIK细胞IFN-γ、TNF-α、TNF-β、穿孔素、颗粒酶A、颗粒酶B、FasL和NKG2D mRNA的表达；酶联免疫法检测培养上清中IFN-γ和TNF-α的表达；Western blot法检测CIK细胞JAK-STAT信号途径的变化.结果在IL-21作用下,CIK细胞的产生由(17.5±4.7)％升至(26.5±2.1)％,对K562细胞的杀伤作用由(22.8±2.8)％升至(44.6±8.3)％,CIK细胞表面IL-21R的表达增加约2倍.CIK细胞IFN-γmRNA的表达由0.3760±0.2358升至0.7786±0.2493,TNF-α mRNA的表达由0.6557±0.1598升至1.3145 ±0.2136,穿孔素mRNA的表达由0.6361 ±0.1457升至0.9831 ±0.1265,颗粒酶B mRNA的表达由0.4084±0.1589升至0.7319±0.1639,FasL mRNA的表达由0.4015±0.2842升至0.7381±0.2568,差异均有统计学意义.CIK细胞表面FasL的表达水平,加IL-21组(0.19％)与未加IL-21组(0.04％)相比,差异有统计学意义.CIK细胞内穿孔素的表达由35.28％升至53.16％,颗粒酶B的表达由43.16％升至78.82％,培养上清中IFN-γ的表达由(25.8 ±6.1)ng/L升至(56.0 ±2.3) ng/L,TNFα的表达由(5.64±0.61) μg/L升至(15.14±0.93) μg/L.STAT3和STAT5b的表达增加.结论人源IL-21可增强外周血来源CIK细胞抗白血病作用,此作用是通过增加其受体的表达,增加穿孔素、颗粒酶B、FasL、IFN-γ和TNFα的表达而实现的,JAK-STAT细胞信号途径的激活是此种作用的机制之一.提示IL-21在增强白血病免疫治疗中具有潜在的临床应用前景.
목적 연구IL-21대외주혈래원적세포인자유도살상세포(CIK세포)항백혈병작용급기궤제.방법 채집분리정상인외주혈단개핵세포,가용세포인자유도배양,용MTT법검측CIK세포적증식능력급기대자혈병세포계K562세포적살상작용；용류식세포술검측CIK세포면역표형급기표면IL-21수체(IL-21R)、FasL、세포내천공소화과립매B적표체；반정량RT-PCR법검측CIK세포IFN-γ、TNF-α、TNF-β、천공소、과립매A、과립매B、FasL화NKG2D mRNA적표체；매련면역법검측배양상청중IFN-γ화TNF-α적표체；Western blot법검측CIK세포JAK-STAT신호도경적변화.결과 재IL-21작용하,CIK세포적산생유(17.5±4.7)％승지(26.5±2.1)％,대K562세포적살상작용유(22.8±2.8)％승지(44.6±8.3)％,CIK세포표면IL-21R적표체증가약2배.CIK세포IFN-γmRNA적표체유0.3760±0.2358승지0.7786±0.2493,TNF-α mRNA적표체유0.6557±0.1598승지1.3145 ±0.2136,천공소mRNA적표체유0.6361 ±0.1457승지0.9831 ±0.1265,과립매B mRNA적표체유0.4084±0.1589승지0.7319±0.1639,FasL mRNA적표체유0.4015±0.2842승지0.7381±0.2568,차이균유통계학의의.CIK세포표면FasL적표체수평,가IL-21조(0.19％)여미가IL-21조(0.04％)상비,차이유통계학의의.CIK세포내천공소적표체유35.28％승지53.16％,과립매B적표체유43.16％승지78.82％,배양상청중IFN-γ적표체유(25.8 ±6.1)ng/L승지(56.0 ±2.3) ng/L,TNFα적표체유(5.64±0.61) μg/L승지(15.14±0.93) μg/L.STAT3화STAT5b적표체증가.결론 인원IL-21가증강외주혈래원CIK세포항백혈병작용,차작용시통과증가기수체적표체,증가천공소、과립매B、FasL、IFN-γ화TNFα적표체이실현적,JAK-STAT세포신호도경적격활시차충작용적궤제지일.제시IL-21재증강백혈병면역치료중구유잠재적림상응용전경.
Objective To explore the effects of humanized interleukin 21 (IL-21) on anti-leukemic activity of cytokine induced killer(CIK) cells derived from peripheral blood(PB) and the mechanism.Methods Mononuclear cells were separated from peripheral blood and cultured with cytokines to induce CIK cells.Proliferation of CIK cells with or without IL-21 stimulation and their cytotoxic activity against K562 cells was measured by MTT method.IL-21 receptor (IL-21R) and immunophenotypes of CIK cells were measured by flow cytometry.The expression of interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),tumor necrosis factor-β(TNF-β),perforin,granzyme A,granzyme B,FasL and NKG2D mRNA were measured by semiquantitative RT-PCR.FasL on the surface of CIK cells and intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry.The concentration of IFN-γ and TNF-α in the cultured supernatant were measured by enzyme immunoassay.JAK-STAT signalling pathway of CIK cells were measured by Westernblot.Results After IL-21 stimulation,the proportion of CIK cells increased from (17.5 ± 4.7) ％ to (26.5 ± 2.1) ％.Cytotoxic activity against K562 cells by CIK cells increased from (22.8 ± 2.8) ％ to (44.6± 8.3)％.The expression of IL-21R increased about 2 folds.The mRNA expression of IFN-γ increased almost 2 folds from (0.3760 ±0.2358) to (0.7786 ± 0.2493),TNF-α increased almost 2 folds from (0.6557 ±0.1598) to (1.3145 ± 0.2136),perforin increased almost 1.5 folds from (0.6361 ±0.1457) to (0.9831 ± 0.1265),granzyme B increased almost 2 folds from (0.4084 ± 0.1589) to (0.7319 ±0.1639),FasL increased almost 2 folds from (0.4015 ±0.2842) to (0.7381 ±0.2568),the expression of granzyme A,TNF-β and NKG2D were similar with control.Flow cytometry analysis showed that the expression of FasL of CIK cells was higher than that of control (0.19％ vs 0.04％),the expression of perforin increased from 35.28％ to 53.16％,and the expression of granzyme B increased from 43.16％ to 78.82％.The concentration of IFN-γ in the culture supernatant increased almost 2 folds from (25.8 ±6.1)ng/L to (56.0 ± 2.3) ng/L,and TNF-α increased almost 3 folds from (5.64 ± 0.61) μg/L to (15.14 ±0.93) μg/L.Western blot showed that the expression of STAT1 and STAT5a had no significant differences,but the expression of STAT3 and STAT5b were higher than that of control.Conclusion Humanized IL-21 could enhance the anti-leukemic activity of CIK cells via increasing IL-21R,perforin,granzyme B,FasL,IFN-γand TNF-α,as well as activating JAK-STAT signaling pathway.These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemic immunotherapy.