Effect of dendritic cells transfected with total RNA of HepG2 cell line using GFP marker in vitro

    ZHANG Liª²Wang, ZHANG Hongª²Mei, LIU Wenª²Chao, PAN Boª²Rong, SI Xiaoª²Ming, REN Jun

    Center of Clinical Oncology£¬Xijing Hospital£¬Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To investigate the feasibility of GFP as a marker to observe the dendritic cells (DCs) transfected with total RNA of tumor cells and the feasibility of the transfected DCs serving as a vaccine for potential immunotherapy. METHODS: Plasmid pGFPª²C3 was transfected into HepG2 stably. Total RNA was extracted from the HepG2ª²GFP using Trizol; DCs were induced by liver cancer patients¬ð peripheral blood mononuclear cells (PBMCs), and transfected with the total RNA. The effect of transfection was observed by a fluorescence microscope, the changes of phenotype were detected by flow cytometry, and the change of ILª²12 secretion in the supernatant of DCs was detected by ELISA assay. The cytotoxic effect of CTLs was assessed by MTT assay. RESULTS: GFP was expressed stably in the HepG2ª²GFP cells that presented green fluorescence under a fluorescence microscope, so did DCs transfected with total HepG2ª²GFP cell RNA. After transfection, the expression of membrane molecules such as CD80 (13.2% to 86.7%), HLAª²DR (38.9% to 97.9%), CD83 (0.9% to 97.1%), CD86 (31.2% to 92.5%) was increased dramatically, ILª²12 secretion in the supernatant was elevated significantly [(61.3¡À8.1) ng/L to (287.4¡À29.3) ng/L, P<0.05]. The CTLs activated by DCs transfected with total HepG2ª²GFP RNA showed a potent specific lysis to HepG2 cells. CONCLUSION: GFP could be used as a marker to observe the effect of transfection of DCs with total tumor cell RNA. DCs transfected with total tumor cell RNA may serve as a promising vaccine of immunotherapy. ......