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目的:将体外转录的癌胚抗原(carcinoembryonic antigen,CEA)mRNA转染入成熟树突状细胞(dendritic cell,DCs),观察其体外诱导的特异性抗肿瘤作用。方法:GM-CSF、IL-4、TNF-α体外诱导成熟DCs并用流式鉴定。构建pcDNA3.1-CEA载体,体外转录为CEA mRNA,电穿孔法将CEA mRNA转染入DCs。流式细胞术检测转染后DCs中CEA蛋白的表达;MTT法检测DCs刺激T细胞增殖能力;LDH法检测DCs体外诱导的CTL的特异性抗肿瘤作用;ELISA法检测诱导的CTL上清中IFN-γ的水平。结果:CEA mRNA转染后DCs细胞内CEA蛋白显著高于对照组(83.32%vs3.34%,P<0.01)。CEA mRNA转染组DCs在效靶比为1∶10时,刺激T细胞增殖作用最强,明显高于未转染组[(19.11±1.89)%vs(15.59±0.70)%,P<0.05]。CEAmRNA转染组DCs能产生CEA特异性的CTL效应,在效靶比为5∶1、10∶1、20∶1和40∶1时杀伤率分别为[(5.42±0.87)%、(14.09±1.13)%、(27.16±0.72)%、(32.49±0.84)%,P<0.01],而未转染组和对照靶细胞均无杀伤作用。转染组DCs诱导的CTL上清中IFN-γ分泌量显著高于未转染组[(141.73±28.61)vs(9.45±4.63)pg/ml,P<0.01]。结论:CEAmRNA转染的成熟DCs体外能产生特异性抗肿瘤作用,为研制CEA RNA-DCs疫苗提供了实验依据。
OBJECTIVE: To transfect carcinoembryonic antigen (CEA) mRNA into mature dendritic cells (DCs) in vitro and to observe its specific antitumor activity. Methods: DCs were induced in vitro by GM-CSF, IL-4 and TNF-α and identified by flow cytometry. The pcDNA3.1-CEA vector was constructed and transcribed into CEA mRNA in vitro. CEA mRNA was transfected into DCs by electroporation. Flow cytometry was used to detect the expression of CEA protein in DCs transfected with DCs. The proliferation of DCs stimulated by DCs was detected by MTT assay. The anti-tumor effect of CTL induced by DCs in vitro was detected by LDH assay. -γ level. Results: The CEA protein of DCs transfected with CEA mRNA was significantly higher than that of the control group (83.32% vs 3.34%, P <0.01). CEA mRNA transfected DCs had the strongest effect on stimulating T cell proliferation when the target ratio was 1:10 [(19.11 ± 1.89)% vs (15.59 ± 0.70)%, P <0.05] . CEAmRNA transfected DCs can produce CEA-specific CTLs with killing rates of (5.42 ± 0.87)%, (14.09 ± 1.13%, (27.16 ± 0.72)%, (32.49 ± 0.84)% respectively, P <0.01], while the untransfected and control target cells had no killing effect. The amount of IFN-γ secretion in CTL supernatant induced by DCs in transfected group was significantly higher than that in non-transfected group [(141.73 ± 28.61) vs (9.45 ± 4.63) pg / ml, P <0.01]. Conclusion: The mature DCs transfected with CEAmRNA can produce specific antitumor activity in vitro and provide experimental evidence for the development of CEA RNA-DCs vaccine.