探讨地西他滨短时刺激对RAK细胞免疫表型及抗癌活性的影响  

Effects on immunophenotype and anticancer activity of RAK cells after short-term stimulation with Dicitabine

作　　者：李洁羽[1] 周智锋[1] 林万松[1] 陈淑萍[1] 叶韵斌(指导)[1] LI Jieyu;ZHOU Zhifeng;LIN Wansong;CHEN Shuping;YE Yunbin(Immuno-Oncology Laboratory of Fujian Medical University Cancer Hospital,Fujian Cancer Hospital,Fujian Key Laboratory of Translational Cancer Medicine,Fuzhou 350014,China)

机构地区：[1]福建医科大学附属肿瘤医院,福建省肿瘤医院肿瘤免疫学研究室,福建省肿瘤转化医学重点实验室,福州350014

出　　处：《中国免疫学杂志》2022年第23期2822-2827,共6页Chinese Journal of Immunology

基　　金：福建省卫健委中青年骨干人才培养项目(2020GGB011);福建省自然科学基金项目(2021J01436)资助。

摘　　要：目的:探讨地西他滨(DAC)对重组人纤维连接蛋白活化杀伤细胞(RAK)增殖、免疫表型及抗肝癌效应的影响。方法:采用RetroNectin、CD3单抗及rhIL-2诱导外周血单个核细胞以培养RAK细胞,培养第1天加入不同浓度的DAC(0、50、500、1 000、2 000、3 000 nmol/L)刺激24 h后,全量换液。培养第4、7、10、12天,采用血球计数仪进行细胞计数,CFSE检测细胞增殖水平;流式细胞术检测不同培养时间各浓度DAC刺激的RAK细胞的免疫表型;Annexin-Ⅴ/PI检测细胞凋亡水平;以CD107a、IFN-γ、穿孔素、颗粒酶-B表达水平评估各组RAK细胞的抗肝癌效应。结果:随着培养时间延长,DAC浓度越高,细胞增殖抑制越明显(P<0.05)。与对照组相比,DAC短时刺激的RAK细胞在培养10 d后,高浓度DAC组(≥1 000 nmol/L)RAK细胞CD4比例显著增加,CD8比例及CD4+和CD8+的中央记忆细胞(TCM)比例呈下降趋势(P<0.05)。培养第12天NKT水平显著升高(P<0.05),随着DAC浓度增高细胞凋亡水平显著升高(P<0.05)。与其他DAC浓度组相比,1 000 nmol/L DAC刺激组CD107a、IFN-γ、穿孔素表达显著升高(P<0.05)。结论:RAK细胞经1 000 nmol/L DAC短时刺激后,可提高CD4、CD4+TCM及NKT比例,增强对肝癌细胞的细胞毒效应。Objective:To investigate effects of Decitabine(DAC)on proliferation,immunophenotype and anti-hepatocellular carcinoma(HCC)of recombinant human fibronectin activated killer cells(RAK).Methods:RAK were generated from peripheral blood mononuclear cells of healthy donors induced by RetroNectin,anti-CD3 monoclonal antibody and rhIL-2. On the first day of culture,different concentrations of DAC(0,50,500,1 000,2 000,3 000 nmol/L)were added to stimulate RAK cells for 24 h,and a fresh culture medium was replaced. At the 4th,7th,10th and 12th day of culture,cells were counted by automatic blood cell counter,and cell proliferation level was measured by CFSE. Immunophenotypes of RAK cells stimulated by DAC at different culture time and concentrations were detected by flow cytometry. Cell apoptosis was detected by Annexin-Ⅴ/PI Kit. CD107a,IFN-γ,perforin,Granzyme B expression levels were used to evaluate anti-HCC effect of RAK cells in each group.Results:DAC could promote early rapid proliferation of RAK cells,and the higher the concentration of DAC,the more obvious the inhibition of cell proliferation was(P<0.05). Compared with control group,after 10 days of culture,proportion of CD4 in RAK cells stimulated by DAC in high-concentration DAC groups(≥1 000 nmol/L)was significantly increased,while proportion of CD8 and expressions of central memory T cell(TCM)of CD4+and CD8+showed a decreasing trend(P<0.05). On the 12th day,level of NKT was increased significantly(P<0.05).Level of apoptosis was increased with the increase of DAC(P<0.05). Compared with other DAC concentration groups,expressions of CD107a,IFN-γ and perforin in RAK cells stimulated by 1 000 nmol/L DAC was significantly increased(P<0.05).Conclusion:RAK cells can increase proportion of CD4,CD4+TCM and NKT cells,and enhance cytotoxic effect on HCC cells after short-term stimulation with 1 000 nmol/L DAC.

关 键 词：地西他滨 重组人纤维连接蛋白 免疫表型 肿瘤免疫 

分 类 号：R392.12[医药卫生—免疫学]