树突状细胞瘤苗对自体肺癌细胞的杀伤作用

Anti-tumor effect of dendritic cell based vaccine against autologous lung cancer cells

目的：利用非小细胞肺癌（nonsmall-cell lung cancer；NSCLC）患者的树突状细胞（dendritic cell，DC）负载自体肿瘤抗原制备DC-CIK（cytokine induced killer cell）瘤苗，观察其在体外对患者自身肿瘤细胞的杀伤作用。方法：提取NSCLC患者外周血单个核细胞（peripheral blood mononuclear cell，PBMC），分别培养成DC与CIK细胞；取患者自身的肿瘤组织，分离、培养出肿瘤细胞，以肿瘤细胞冻融物、坏死肿瘤细胞、正常生长的肿瘤细胞为抗原，分别负载DC，并经OK-432激活，流式细胞仪检测DC表面分子的表达；将各组DC与CIK细胞共培养，MTT法测定DC-CIK对自身肿瘤细胞的杀伤作用。结果：负载肿瘤细胞冻融抗原的DC，其象征成熟的表面分子CD1a、CD80、CD83、HLR-DR表达分别达到85．1±2．7、80．0±4．4、75．4±5．3、80．5±7．8，明显高于负载坏死肿瘤细胞抗原的DC和负载正常生长肿瘤细胞的DC（P〈0．01）；而负载正常生长肿瘤细胞的DC，其上述分子的表达受到抑制。各组DC与CIK共培养后，负载肿瘤细胞冻融抗原的DC可明显刺激CIK的增殖，使CIK细胞增加3—6倍，并使CIK细胞中CD3’CD56’细胞的含量增至（65．8±15．5）％，同时加强CIK对自身肿瘤细胞的杀伤力（P〈0．01）。结论：NSCLC患者PBMC来源的DC在负载自身肿瘤细胞冻融抗原后，可分化为成熟DC；该DC可促进CIK细胞的增殖、提高DC-CIK瘤苗对自身肿瘤细胞的杀伤作用。

Objective： To prepare dendritic cell （DC） -cytokine induced killer （CIK） tumor vaccine using DCs of patients with nonsmall-cell lung cancer （NSCLC） harboring autologous tumor antigens and to observe its inhibitory effect on autologous tumor cells in vitro. Methods： Peripheral blood mononuclear cells （PBMCs） extracted from NSCLC patients were cultured into DCs and CIKs in presence of different cytokines. Cancer cells from the patients were cultured in vitro and were used to prepare different tumor antigens ： tumor cell lysates, necrotic tumor cells and normally-grown tumor cells. The DCs were stimulated by OK-432 and the phenotypes of the DCs were analyzed by flow cytometer. Then the DCs were co-cultured with CIKs. The anti-tumor effect of DC-CIK was evaluated by MTT assay. Results： Compared with the DCs harboring the antigens of necrotic tumor cells and normally-grown tumor cells, DCs pulsed with tumor cell lysates had significantly higher expression of surface molecules such as CD1 a （85.1±2.7 ）, CD80 （ 80.0±4.4 ）, CD83 （75.4±5.3） , and HLA-DR（80.5±7.8, all P 〈0.01 ）. The expression of the above molecules was inhibited in DCs harboring antigen of normal grown tumor cells. DCs harboring tumor cell lysates as antigen promoted the proliferation of CIKs by 3-6 times and increased the proportion of CD3^＋ CD56^＋ cells in the CIKs to （65.8±15.5 ）% ; meanwhile, it enhanced the anti-tumor effect of CIK against autologous tumor cells. Conclusion： DCs derived from PBMCs of NSCLC patients harboring the frozen-melted autologous tumor cell antigens can differentiate into mature DCs, which can facilitate the proliferation of CIK cells and enhance the effect of DC-CIK tumor vaccine against tumor cells.