负载肺癌全抗原的自体树突状细胞诱导T细胞反应的体外研究

In vitro study of T cell responses induced by lung cancer's whole antigen pulsed with autologous dendritic cells

目的在体外研究负载肺癌全抗原的自体树突状细胞（DCs）诱导的T细胞增殖及细胞毒作用。方法肺癌组织、正常肺组织和外周血新鲜标本均取自肺鳞状细胞癌并行手术治疗的7例患者,肺癌组织和正常肺组织制备为单细胞悬液。自外周血分离单核细胞并诱导DCs,尼龙毛柱法分离T细胞。冻融法制备自体肺癌细胞裂解液,并刺激DCs,制备负载自体肺癌全抗原的DCs。MTS法检测负载肺癌全抗原的DCs对T细胞增殖的影响;流式细胞术测定DCs表型及负载肺癌全抗原的DCs对T细胞细胞毒作用的影响。结果外周血单核细胞经GM-CSF、IL-4和TNF-α诱导培养后,在细胞膜上出现大量的树突状突起,表面分子CD14表达显著降低（t=20.157,P〈0.01）,CD1a、CD11c、CD80、CD83和HLA-DR表达明显增加（t=8.927,3.042,14.311,18.537,5.612,P〈0.05,P〈0.01）,获得负载肺癌全抗原的成熟DCs;其能刺激自体T细胞增殖,不同患者刺激指数不同为6.98-39.15（21.56±4.38）,而且DC/T比率越高,刺激指数越高,T细胞增殖反应能力越强（t=31.203,19.550,P〈0.01）;负载肺癌细胞全抗原的DCs能诱导自体T细胞的细胞毒作用,且T细胞对自体肿瘤细胞的杀伤率为（56.52±2.75）%;对正常细胞的杀伤率平均为（5.31±1.37）%,明显低于对自体肿瘤细胞杀伤率（t=16.429,P〈0.01）。对K562细胞杀伤率为（9.71±2.46）%,明显低于对自体肿瘤细胞杀伤率（t=25.010,P〈0.01）。结论负载肺癌全抗原的DCs能诱导肺癌患者自体T细胞增殖及细胞毒作用,且体外可有力的杀伤肿瘤细胞,而几乎不杀伤正常细胞。

Objective To investigate the load cell lung cancer antigen autologous dendritic （ DCs ）-induced T cell proliferation and cytotoxicity in vitro. Methods Lung cancer tissue, normal lung tissue and peripheral blood of fresh speci-mens were taken from 7 patients with squamous cell carcinoma who were treated with operation, normal lung tissue and lung cancer were prepared for single cell suspension. From the peripheral blood, mononuclear cells isolated and induced DCs;ny-lon wool columns were used to isolate the T cells. Using frozen melt method to prepare the autologous lung cancer cell lysates liquid, and stimulate the DCs, preparation of loaded with autologous lung cancer antigen DCs. Using MTS method to detect DCs complete antigen load of lung cancer on T cell proliferation;flow cytometry determination of DCs phenotype and the effect of load DCs complete antigen of lung cancer on T cell cytotoxicity. Results Peripheral blood mononuclear cells through GM-CSF, IL-4 and TNF-α’ s induce, a large number of dendritic protrusions, surface molecules CD14 expression was significantly lower （ t =20. 157, P 〈0. 01）, CD1a, CD11c, CD80, CD83 and HLA-DR expression was significantly increased （ t =8. 927, t =3. 042, t =14. 311, t =18. 537, t =5. 612, P 〈0. 05, P 〈0. 01）, mature DCs which was obtain from whole load of lung cancer antigen can stimulate autologous T cell proliferation, different patients revealed different stimulation index：6. 98-39. 15 （21. 56 ± 4. 38）, and the higher the DC/T ratio, the higher the stimulation index, the stronger of T cells ability to respond to the proliferation （ t =31. 203, t =19. 550, P 〈0. 01）;DCs of load lung cancer cell whole antigen can induce cytotoxicity of autologous T cells,and T cells to autologous tumor cell killing rate was（56. 52 ± 2. 75）%;normal cell average killing rate was （5. 31 ± 1. 37）%, significantly lower than for the autologous tumor cell killing rate （ t =16. 429, P 〈0. 01）. The killing rate of K562 cell was （9. 71 ± 2. 46）%, which is significantly lower than the killing rate of autologous tumor cells （t = 25. 010, P 〈0. 01）. Conclusion Load lung cancer complete antigen’s DCs can induce lung cancer pa-tients’ autologous T cell proliferation and cytotoxicity, and can effectively kill tumor cells in vitro, and normal cells were not killed.