两种抗原致敏方式负载树突状细胞疫苗对结直肠癌细胞株的体外杀伤作用

Effects of two different antigen-loading methods on the activity of dendritic cell vaccine for colorectal carcinoma cell inhibition in vitro

目的比较两种抗原负载方式对树突状细胞(DCs)疫苗的影响。方法抽取HLA表型为A11的健康志愿者外周血,分离单核细胞,体外培养。通过反复冻融LoVo细胞,提取肿瘤细胞裂解物或者使用含CEA片断的重组腺相关病毒转染未成熟DCs,诱导特异性细胞毒性T细胞。检测体外培养的DCs和CTL活性,并使用MTT法检测两组CTL对LoVo细胞的杀伤作用。结果两种方式均可培养的成熟DCs,诱导的CTL细胞分泌IFN-γ有所增加;转染后DCs诱导特异性CTL可有效识别并杀伤HLA-A11阳性的LoVo细胞,腺相关病毒提呈抗原制备的DCs疫苗对LoVo细胞的杀伤作用明显高于肿瘤细胞裂解物的抗原负载方式。结论两种抗原提呈方式均可培养出成熟的DCs,不明显改变DCs表型和刺激淋巴细胞增殖、分化功能,并可诱导自体CTL增殖。使用腺相关病毒转染DCs的方式明显优于肿瘤细胞裂解物的抗原负载方式。

Objective To observe the changes in the activity of dendritic cells （DCs） after carcino-embryonic antigen （CEA） gene transfection mediated by recombinant adeno-associated virus type2 （rAAV） and tumor cell lysate. Methods Immature DCs isolated from peripheral blood monocytes ofHLA-A11-positive healthy volunteers were infected with the rAAV carrying CEA gene or loaded with tumor cell lysate. The surface markers of the DCs such as CD40, CD 1α, and CD86 were analyzed by flow cytometry. Interleukin-12 （1L-12） in the supematants of DCs and interferon-γ （IFN-γ） released by the cytotoxic T lymphocytes （CTLs） were determined by ELISA detection kit. The specific killing activity of CTL against LoVo cells was assessed by MTT assay. Results The DCs following antigen loading with the two methods both highly expressed CD40, CD86 and IL-12, and induced specific CTL that specifically recognized and killed LoVo cells, but the killing effect resulting from rAAV infection of the DCs was much better than that induced by tumor cell lysate loading. Conclusion Both methods of antigen loading can induce mature DCs from peripheral blood monocyte cells, but rAAV infection of the DCs can be more effective than tumor cells lysate loading. DCs infected with rAAV may have the potential to serve as an adjuvant immunotherapy for patients with colorectal carcinoma.