AR-A014418对γδT细胞增殖、凋亡及杀伤功能的影响
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摘要
目的:观察糖原合酶激酶-3β(GSK-3β)选择性抑制剂AR-A014418对体外扩增的γδT细胞的细胞活性包括增殖、凋亡和杀伤功能的影响。方法:采用人淋巴细胞分离液分离健康人外周血中单个核细胞,在γδT细胞完全培养基中培养获得γδT细胞。用不同浓度的AR-A014418诱导培养γδT细胞72 h后,用流式细胞术检测各诱导处理组γδT细胞的增殖、凋亡和杀伤能力;用Western blot检测γδT细胞凋亡相关蛋白Bcl-2、Bax和Caspase-3表达情况。结果:AR-A014418浓度在0. 3～10μmol/L时能促进γδT细胞的增殖并抑制其凋亡。同时促凋亡蛋白Cleaved caspase-3和Bax表达逐渐减弱,而抗凋亡蛋白Bcl-2表达逐渐增强。尤其是在3μmol/L时,γδT细胞增殖活性最强,而且体外杀伤人肝癌HepG2细胞和人胃癌SGC-7901细胞活性也显著提高。结论:GSK-3β抑制剂AR-A014418在3μmol/L浓度时,可以抑制体外扩增γδT细胞的凋亡,并能增强其体外增殖和杀伤肿瘤活性,提示AR-A014418具有一定的免疫调节功能,为今后用于体外扩增γδT细胞提供实验依据。
Objective: To investigate the effect of glycogen synthase kinase-3β selective inhibitor AR-A014418 on human γδT cells growth,apoptosis and killing function in vitro. Methods: Peripheral blood mononuclear cell( PBMCs) was separated using FicollHypaque gradient centrifugation and cultivated in γδT complete medium in vitro. After co-cultured with various concentrations of ARA014418 for 72 h,the proliferation,apoptosis and killing function in each group were determined by flow cytometric analysis. The protein expression was examined by Western blot analysis. Results: AR-A014418 at 0. 3-10 μmol/L could promote the growth of γδT cells and enhance the cytotoxic activity of γδT cells to Hep G2 cells and SGC-7901,especially at 3 μmol/L for 72 h. Furthermore,ARA014418 could upregulate the expression of the anti-apoptotic protein Bcl-2 and inhibition of pro-apoptotic protein cleaved caspase-3 and Bax expression. Conclusion: AR-A014418 at indicated concentrations can promote the γδT cells growth and enhance the cytotoxic activity of γδT cells to Hep G2 and SGC-7901 cells. These findings suggest that AR-A014418 can be a useful complementary agent for improving γδT cell-based immunotherapy.
Objective: To investigate the effect of glycogen synthase kinase-3β selective inhibitor AR-A014418 on human γδT cells growth,apoptosis and killing function in vitro. Methods: Peripheral blood mononuclear cell( PBMCs) was separated using FicollHypaque gradient centrifugation and cultivated in γδT complete medium in vitro. After co-cultured with various concentrations of ARA014418 for 72 h,the proliferation,apoptosis and killing function in each group were determined by flow cytometric analysis. The protein expression was examined by Western blot analysis. Results: AR-A014418 at 0. 3-10 μmol/L could promote the growth of γδT cells and enhance the cytotoxic activity of γδT cells to Hep G2 cells and SGC-7901,especially at 3 μmol/L for 72 h. Furthermore,ARA014418 could upregulate the expression of the anti-apoptotic protein Bcl-2 and inhibition of pro-apoptotic protein cleaved caspase-3 and Bax expression. Conclusion: AR-A014418 at indicated concentrations can promote the γδT cells growth and enhance the cytotoxic activity of γδT cells to Hep G2 and SGC-7901 cells. These findings suggest that AR-A014418 can be a useful complementary agent for improving γδT cell-based immunotherapy.
引文
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[3]Muzny DM,Bainbridge MN,Chang K,et al.Comprehensive molecular characterization of human colon and rectal cancer[J].Nature,2012,487(7407):330-337.
[4]Sawa M,Masuda M,Yamada T.Targeting the Wnt signaling pathway in colorectal cancer[J].Expert Opin Ther Targets,2016,20(4):419-429.
[5]Gattinoni L,Lugli E,Ji Y,et al.A human memory T cell subset with stem cell-like properties[J].Nat Med,2011,17(10):1290-1297.
[6]Sabatino M,Hu J,Sommariva M,et al.Generation of clinical-grade CD19-specific CAR-modified CD8+memory stem cells for the treatment of human B-cell malignancies[J].Blood,2016,128(4)519-528.
[7]陈永强,郑璐,刘军权,等.糖原合酶激酶-3β抑制剂TWS119对γδT细胞增殖及表型的影响[J].中国免疫学杂志,2015,31(7):66-70.Chen YQ,Zheng L,Liu JQ,et al.Effect of glycogen synthase kinase-3βinhibitor TWS119 on proliferation and phenotypic characteristics of humanγδT cells in vitro[J].Chin J Immunol,2015,31(7):66-70.
[8]陈永强,郑璐,刘军权,等.TWS119对人胃癌SGC-7901细胞和γδT细胞活性影响及机制研究[J].中国药学杂志,2016,51(5):373-378.Chen YQ,Zheng L,Liu JQ,et al.Effect of TWS119 on the Activities of Human SGC-7901 andγδT cells in vitro and its mechanism[J]Chin Pharm J,2016,51(5):373-378.
[9]Bhat R,Xue Y,Berg S,et al.Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418[J].J Biol Chem,2003,278(46):45937-45945.
[10]Beetz S,Wesch D,Marischen L,et al.Innate immune functions of human gammadelta T cells[J].Immunobiology,2008,213:173-182.
[11]Hannani D,Ma Y,Yamazaki T,et al.HarnessingγδT cells in anticancer immunotherapy[J].Trends Immunol,2012,33(5):199-206.
[12]Kobayashi H,Tanaka Y.γδT cell immunotherapy-A review[J].Pharmaceuticals(Basel),2015,8(1):40-61.
[13]Li W,Kubo S,Okuda A,et al.Effect of IL-18 on expansion of gammadelta T cells stimulated by zoledronate and IL-2[J].J Immunother,2010,33(3):287-296.
[14]郑璐,陈永强,刘军权,等.槲皮苷对人γδT细胞增殖及杀伤功能的影响[J].中华微生物和免疫学杂志,2014,34(6):437-441.Zheng L,Chen YQ,Liu JQ,et al.Effects of quercitrin on the proliferation and the cytotoxicity of humanγδT cells[J].Chin J Microbiol Immunol,2014,34(6):437-441.
[15]Zhou ZH,Chen FX,Xu WR,et al.Enhancement effect of dihydroartemisinin on humanγδT cell proliferation and killing pancreatic cancer cells[J].Int Immunopharmacol,2013,17(3):850-857.
[16]陈玲,吕小婷,陈复兴,等.帕米膦酸二钠与唑来膦酸对体外培养人γδT细胞的杀伤功能影响比较[J].免疫学杂志,2016,32(12):1039-1042.Chen L,Lv XT,Chen FX,et al.Comparison of the effect onγδTcell killing activity between zoledronic acid and pamidronate disodium[J].Immunol J,2016,32(12):1039-1042.
[17]Gattinoni L,Klebanoff CA,Restifo NP.Pharmacologic induction of CD8+T cell memory:better living through chemistry[J].Sci Transl Med,2009,1(11):11-12.