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目的观察EB病毒(Epstein-Barr virus,EBV)-潜伏膜蛋白2A(latent membrane protein2A,LMP2A)转染人树突状细胞(dendritic cell,DC)诱导特异性细胞毒性T细胞(cytotoxicity Tlymphocyte,CTL)的体外生物学特性;建立表达EBV-LMP2A的裸鼠鼻咽癌动物模型,探讨LMP2A特异性CTL在荷瘤鼠体内的抗肿瘤效应。方法分离人外周血单核细胞(pripheral blood mononuclearcell,PBMC),以粒细胞-巨噬细胞集落刺激因子(granulocyte-monocyte colony stimulating factor,GM-CSF)、白细胞介素4(interleukin-4,IL-4)及肿瘤坏死因子(tumor necrosis factor-α,TNF-α)诱导培养获得DC,荧光激活细胞分选仪(fluorescence activated cell sorter,FACS)检测成熟DC的表面分子表达。用LMP2A重组腺病毒转染成熟DC,将转染DC与自体PBMC混合培养,在白细胞介素2(interleukin-2,IL-2)作用下诱导针对LMP2A的特异性CTL,FACS检测CTL群体中阳性细胞的组成。将鼻咽癌CNE细胞接种BALB/c裸鼠,建立鼻咽癌动物模型;在裸鼠肿瘤局部注射LMP2A特异性CTL,观察治疗后移植瘤生长情况及病理变化。结果人外周血PBMC体外在GM-CSF、IL-4、TNF-α诱导下获得典型形态及表型特征的成熟DC。FACS检测表明,以LMP2A重组腺病毒转染DC诱导的CTL细胞群体以CD4、CD8阳性细胞组成为主。在接种CNE细胞3周后建立表达EBV-LMP2A的裸鼠鼻咽癌动物模型;动物体内实验表明注射CTL的裸鼠肿瘤生长缓慢,体积明显小于对照组(P<0.01);病理检查显示肿瘤局部发生液化性坏死并有淋巴细胞浸润。结论人外周血单核细胞体外经细胞因子诱导,可生成具典型特征的成熟DC。利用LMP2A重组腺病毒将LMP2A基因转入DC,可在同一个体体外诱导出针对LMP2A的特异性CTL。CNE细胞接种裸鼠,可成功构建鼻咽癌动物模型;LMP2A特异性CTL在动物体内可明显抑制鼻咽癌肿瘤的生长,发挥有效的抗肿瘤作用。
Objective To observe the effects of Epstein-Barr virus (EBV) and latent membrane protein 2A (LMP2A) on the expression of cytotoxic T lymphocyte (CTL) in human dendritic cells (DCs) In vitro. To establish an animal model of nasopharyngeal carcinoma expressing EBV-LMP2A in nude mice and investigate the antitumor effect of LMP2A-specific CTL in tumor-bearing mice. Methods Human peripheral blood mononuclearcells (PBMCs) were isolated from human peripheral blood mononuclear cells (PBMCs) and transfected with granulocyte-monocyte colony stimulating factor (GM-CSF), interleukin-4 (IL- DCs were harvested from tumor necrosis factor-α (TNF-α, tumor necrosis factor-α and tumor necrosis factor-α), and the surface molecules of mature DCs were detected by fluorescence activated cell sorter (FACS). Mature DCs were transfected with LMP2A recombinant adenovirus and transfected DCs were mixed with autologous PBMCs to induce specific CTLs against LMP2A under the action of interleukin-2 (IL-2). The CTL colonies were positive by FACS The composition of the cells. Nasopharyngeal carcinoma CNE cells were inoculated BALB / c nude mice to establish nasopharyngeal carcinoma animal model; local injection of LMP2A specific CTL in nude mice tumor growth and pathological changes after treatment. Results Peripheral blood PBMCs were induced by GM-CSF, IL-4 and TNF-α to produce mature DCs with typical morphological and phenotypic characteristics. FACS analysis showed that the population of CTL cells induced by DCs transfected with LMP2A recombinant adenovirus mainly consisted of CD4 and CD8 positive cells. Three weeks after inoculation of CNE cells, EBV-LMP2A-expressing NPC mice were established. The animal experiments in vivo showed that CTL-injected nude mice grew slowly and the volume was significantly smaller than that of the control group (P <0.01). Pathological examination showed that tumor localized Liquefaction necrosis and lymphocytic infiltration occurred. Conclusion Human peripheral blood mononuclear cells induced by cytokines in vitro can produce mature DCs with typical features. Using LMP2A recombinant adenovirus to transfer LMP2A gene into DC, we can induce specific CTL against LMP2A in the same individual. CNE cells inoculated nude mice successfully constructed nasopharyngeal carcinoma animal model; LMP2A specific CTL in vivo can significantly inhibit the growth of nasopharyngeal carcinoma tumors, to play an effective antitumor effect.