【摘要】 目的:检测绿荧光蛋白基因(EGFP)质粒表达载体pACCMV-EGFP和pACTERT-EGFP对SACC-83的转染效率及瞬时表达,为应用这两种启动子诱导促凋亡基因转染SACC-83以发挥治疗作用提供依据。方法:实验于2004-07/2005-05在吉林大学口腔医学院口腔生物实验室完成。①在体外扩增并酶切鉴定pACCMV-EGFP和pACTERT-EGFP质粒。②以脂质体介导法转染pACCMV-EGFP和pACTERT-EGFP进入SACC-83细胞,根据质粒和脂质体比例不同分别转染6组,1组:1g/4L;2组:1g/8L;3组:1g/12L;4组:2g/4L;5组:2g/8L;6组:2g/12L。③应用荧光显微镜观察各组转染效率及瞬时表达情况,并进行统计学分析。结果:①质粒的检测及鉴定:质粒pACTERT-EGFP和pACCMV-EGFP经转化、酶切电泳证实,均出现约800bp的片段,与原作者提供的数据一致。②荧光显微镜观察:绿荧光蛋白在基因转染24h后开始表达,48～72h最高,1周后表达逐渐减弱。③荧光细胞数的统计学分析:对不同浓度质粒与脂质体转染72h的荧光细胞数进行方差分析,质粒与脂质体比例为1g/8L和2g/8L组荧光细胞数显著高于其余各组,其差异具有统计学意义,说明转染效率与脂质体和质粒的比例有关。结论:按照合适的质粒和脂质体比例,pACCMV-EGFP和pACTERT-EGFP转染SACC-83的效率可以达到30%,是转染SACC-83较为理想的瞬时表达载体。更多还原

【Abstract】 AIM: To determine the optimized condition under which pro-apoptotic gene expression vectors with CMV and hTERT promoter will be constructed to transfect salivary adenoid cystic carcinoma cells (SACC-83), plasmid vectors coding enhanced green fluorescence protein (EGFP) gene pACCMV-EGFP and pACTERT-EGFP were transfected into SACC-83. The transfection efficiency and transient expression were subsequently tested. METHODS: The experiment was performed in Oral-biology Lab, Stomatology School, Jilin University from July 2004 to May 2005. ①pACCMV-EGFP and pACTERT-EGFP were amplified and tested by an enzyme cutting technique in vitro; ②pACCMV-EGFP and pACTERT-EGFP were transferred into SACC-83 by means of lipofectamine media methods. There were 6 groups for each plasmid according to various ratios of plasmid to lipofectamine, group 1: 1 g/4 L;group 2:1 g/8 L;group 3:1 g/12 L;group 4:2 g/4 L;group 5:2 g/8 L;group 6:2 g/12 L. ③Transfer efficiency and transient expression were evaluated by fluorescent microscopy. Statistical analysis was conducted. RESULTS: ①Determination and identification of plasmid: After transformation, pACCMV-EGFP and pACTERT-EGFP were cut and 800 bp fragments were observed through electrophoreses, which conformed to plasmid map provided by the original author. ②Observed with fluorescent microscope: The expression of EGFP began in 24 hours after transferring, reached maximum in 48-72 hours and decreased after one week. ③Statistical analysis of fluorescent cell counting: Differences of fluorescent cell number in 72 hours after transferring among the experiment groups were assessed by analysis of variance (ANOVA). Fluorescent cell number of the group with the ratio of plasmid to lipofectamine (1 g/8 L and 2 g/8 L) was significantly higher than that of the rest groups, and the difference had statistical significance, which showed that transfer efficiency was correlated with the ratio of plasmid to lipofectamine. CONCLUSION: The efficiency of transferring pACCMV-EGFP and pACTERT-EGFP into SACC-83 can achieve to 30% with proper ratio of plasmid to lipofactamine. pACCMV-EGFP and pACTERT-EGFP are ideal transient expression vectors for SACC-83 gene transference.更多还原