以GFP为标记的肝癌细胞RNA转染树突状细胞的体外效应  

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

作　　者：张利旺[1] 张红梅[1] 刘文超[1] 潘伯荣[1] 斯晓明[1] 任军[1] 

机构地区：[1]第四军医大学西京医院肿瘤中心,陕西西安710033

出　　处：《第四军医大学学报》2006年第11期1046-1048,共3页Journal of the Fourth Military Medical University

摘　　要：目的:探讨GFP作为标记观察肝癌细胞RNA转染DCs效果的可行性及肿瘤细胞RNA转染DCs制备疫苗的可行性.方法:绿色荧光蛋白质粒载体pGFPC3稳定转染肝癌细胞HepG2,Trizol法提取筛选后细胞HepG2GFP总RNA;分离肝癌患者外周血单核细胞体外诱导DCs细胞,总RNA转染DCs,荧光显微镜下观察转染效果,流式细胞仪检测转染前后DCs表型变化;ELISA法检测转染前后上清中IL12变化情况;MTT法检测效应细胞对靶细胞的杀伤率.结果:pGFPC3稳定转染肝癌细胞HepG2后可得到稳定表达GFP的细胞HepG2GFP,荧光显微镜下呈绿色荧光;总RNA转染的DCs荧光显微镜下呈绿色荧光,CD80(13.2%上升至86.7%),HLADR(38.9%上升至97.9%),CD83(0.9%上升至97.1%),CD86(31.2%上升至92.5%)表达明显升高,上清IL12分泌量ng/L显著增高(61.3±8.1→287.4±29.3,P<0.05);诱导的CTL能够对肝癌细胞株HepG2起特异性杀伤作用.结论:GFP可以作为肿瘤细胞RNA转染树突状细胞的观察标记,肿瘤细胞RNA转染DCs可作为一种有效的肿瘤疫苗.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 pGFP-C3 was transfected into HepG2 stably. Total RNA was extracted from the HepG2-GFP using Trizol; DCs were induced by liver cancer patients＇ 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 IL-12 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 HepG2-GFP cells that presented green fluorescence under a fluorescence microscope, so did DCs transfected with total HepG2- GFP cell RNA. After transfection, the expression of membrane molecules such as CD80 （13. 2% to 86. 7%）, HLA-DR （38.9% to 97.9%）, CD83 （0. 9% to 97. 1%）, CD86 （31.2% to 92.5% ） was increased dramatically, IL-12 secretion in the supernatant was elevated significantly [ （61.3 ±8.1 ） ng/L to （287.4 ± 29.3 ） ng/L, P 〈 0.05 ]. The CTLs activated by DCs transfected with total HepG2-GFP 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.

关 键 词：树突状细胞 绿色荧光蛋白 RNA 转染 

分 类 号：R739.41[医药卫生—肿瘤]