绿色荧光蛋白报道基因转染鼻咽癌细胞系CNE1后的表达:用做转染报道基因的可行性鉴定  

Expression of a "humanized" green fluorescent protein reporter gene in nasopharyngeal carcinoma cell line CNE1:Feasibility evaluation as transfected reporter gene

作　　者：唐泽立[1] 苏维勇 陈小毅[1] 陈燕[1] 谢玲[1] 

机构地区：[1]广东医学院病理学教研室,广东省湛江市524023 [2]湛江市霞山区霞湖医院,广东省湛江市524006

出　　处：《中国组织工程研究与临床康复》2008年第28期5486-5489,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:将经过"人类化"改造的绿色荧光蛋白(green fluorescent protein,GFP)基因质粒导入鼻咽癌CNE1细胞系内,观察GFP报道基因转染鼻咽癌细胞系CNE1后的表达情况。方法:实验于2004-07/2007-07在广东医学院病理学实验室完成。将带有GFP报道基因的质粒PAT-GFP及PAT-GFP-LMP1在电容960μF,280V条件下电导转染到鼻咽癌CNE1细胞系中,用选择培养液(嘌呤霉素筛选)继续换液培养直至长出抗性细胞克隆,胰酶消化收集细胞。在488nm光激发下,用荧光显微镜检测及流式细胞仪观察转染细胞在活的、固定后或裸鼠体内移植后3组细胞的GFP的表达情况并计算转染效率,并用免疫组化法及Western blot法观察插入目的基因LMP1的表达情况。结果:温度在28～32℃的条件下活细胞有GFP基因稳定而持续地表达,但表达较低,表达率为(16.20±1.70)%,且目的基因LMP1的插入明显影响GFP基因的表达,表达率为(3.48±0.28)%,而在固定后或裸小鼠体内移植后均无表达;目的基因LMP1则能稳定而持续地表达。结论:绿色荧光蛋白报道基因GFP在CNE1细胞系中能稳定而持续地表达,且不影响插入目的基因的表达,可用做转染报道基因。AIM： A ＂humanized＂ green fluorescent protein （GFP） gene plasmid is introduced into nasopharyngeal carcinoma cell line CNE1. This study observed the expression of a ＂humanized＂ green fluorescent protein （GFP） reporter gene in nasopharyngeal carcinoma cell line CNE1. METHODS： Experiments were performed at the Laboratory of Pathology, Guangdong Medical College from July 2004 to July 2007. The plasmid PAT-GFP and PAT-GFP-LMP1 carried the GFP reporter gene were transfected into a nasopharyngeal carcinoma cell line CNEI by electroporation （960 μF, 280 V）, and cultured with selective media （stillomycim screening） until cell clones appeared, and then cells were collected after digesting with trypsin. The expression of GFP was detected and transfected efficiency was calculated in the living cells, the fixed transfected cells and the implanted tumor cells in nude mice by fluorescent microscope and flow cytometry. The expression of LMP1 in transfected cells was detected by iinmunohistochemical method and Western blot. RESULTS： GFP gene could be detected in intact living cells but not in fixed cells and the implanted tumor cells at 28-32℃, the expressed fluorescence was persistent and stable, but the rate of expression was low as （16.20± 1.70）% and could be decreased significantly as （3.48±0.28）% by the inserting of the target gene LMP1. LMP1 was expressed persistently and stably. CONCLUSION： GFP gene can be expressed persistently and stably in CNE1 cells. The expression of target gene LMP1 is not affected by GFP gene. The GFP gene can be used as a marker for gene transfection.

关 键 词：绿色荧光蛋白 标记基因 鼻咽肿瘤 CNE1细胞 

分 类 号：R739.6[医药卫生—肿瘤]