LMPs特异性T淋巴细胞对鼻咽癌细胞的杀伤作用  

The cytotoxic effect of LMPs specific T lymphocytes on nasopharyngeal carcinoma cells

作　　者：李文杰[1] 唐小军[2] 熊四平[2] 陈志[1] 蒋帅[1] 冯振卿[2] 朱进[3] 陈仁杰[1] 

机构地区：[1]南京医科大学第二附属医院耳鼻咽喉科,江苏南京210011 [2]南京医科大学卫生部抗体技术重点实验室,江苏南京210029 [3]南京军区军事医学研究所,江苏南京210002

出　　处：《南京医科大学学报（自然科学版）》2014年第6期705-710,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省临床医学科技专项(BL2013038);江苏省普通高校研究生科研创新计划(CXZZ12_0563)

摘　　要：目的:研究树突状细胞负载EB病毒潜伏膜蛋白(latent membrane proteins,LMPs)介导生成的LMPs特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)LMPs-CTL的生物学特性,观察其对LMPs阳性鼻咽癌细胞SUNE的杀伤作用。方法:用淋巴细胞分离液分离人外周血单核细胞,采用贴壁分离及白细胞介素(interleukin,IL)-4、粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)等细胞因子诱导成熟树突状细胞(dendritic cell,DC),通过DC细胞负载LMPs多肽抗原并递呈给同源T淋巴细胞,制备LMPs-CTL。CCK8法检测LMPs-CTL对SUNE细胞的杀伤作用;ELISA和羟基荧光素二醋酸盐琥珀酰亚胺脂(carboxy fluoroscein succinimidyl ester,CFSE)染色法检测LMPs-CTL分泌IFN-γ及其增殖。结果:LMPsCTL对SUNE细胞的杀伤率在12 h和24 h分别为(43.47±1.93)%和(77.15±3.18)%,显著高于对照组的(11.45±3.06)%和(24.27±13.20)%(P<0.05)。SUNE细胞刺激后,LMPs-CTL增殖能力较对照组有明显增强,IFN-γ分泌量达到(613.40±121.77)pg/ml,显著高于对照组(86.90±3.70)pg/ml(P<0.05)。结论:通过DC细胞负载LMPs混合多肽可诱导生成LMPsCTL,对LMPs阳性鼻咽癌细胞有较强的杀伤作用。Objective：To study the biological characteristics of cytotoxic T lymphocyte（CTL） originated from dendritic cells（DC）loaded with the Epstein-Birr virus latent membrane proteins（LMPs）,and to detect the cytotoxic effect of LMPs-CTL on the LMPs positive nasopharyngeal carcinoma cell SUNE.Methods：The mononuclear cells were isolated from human peripheral blood by lymphocyte separation medium.The adherent method was performed.Cytokines of IL-4 and GM-CSF were performed to induce mature DC.Loaded with LMPs polypeptide antigen,the mature DC presented antigen to autologous T lymphocyte for preparation of LMPsCTL.The cytotoxic effect of LMPs-CTL on SUNE cells was detected by the CCK8 method.The secretion of interferon-γ（IFN-γ） of LMPs-CTL and cell proliferation was detected by ELISA and carboxy fluoroscein succinimidyl ester（CFSE）,respectively.Results：The killing efficiency of LMPs-CTL for SUNE was（43.47 ± 1.93）% in 12h and（77.15 ± 3.18）% in 24 h,which were higher than those in the control group[（11.45 ± 3.06）% and（24.27 ± 13.2）%（P〈 0.05）].The ability of LMPs-CTL cell proliferation simulated with SUNE cells was stronger than that in the control group.The IFN-γ secretion level secreted by LMPs-CTL was（613.40 ± 121.77）pg/ml,which was significantly higher than that secreted by the control group（86.90 ± 3.70） pg/ml（P〈 0.05）.Conclusion：LMPs specific CTL can be successfully induced by mature DC and possess preferable specific killing activity on LMPs positive nasopharyngeal carcinoma cells.

关 键 词：EB病毒 潜伏膜蛋白 树突状细胞 细胞毒性T细胞 鼻咽癌 细胞免疫治疗 

分 类 号：R739.6[医药卫生—肿瘤]