【摘要】 目的:优化聚酰胺-胺型树枝状高聚物(PAMAM-D)纳米载体介导增强型绿色荧光蛋白(EGFP)基因转染人舌鳞癌Tca8113细胞的条件,以获得最佳的转染效率和细胞内表达。方法:采用不同的质粒DNA浓度、PAMAM-D代数、PAMAM-D∶DNA质量比以及转染时间等参数,在激光共聚焦显微镜下,观察PAMAM-D介导pEGFP-N1体外转染Tca8113细胞,检测转染效率,并应用单因素方差分析等统计学方法进行比较分析;结合细胞生长和荧光蛋白的表达情况,进一步评估和优化转染条件。结果:1.0μg质粒DNA与2.0μlG5PAMAM-D形成复合物,转染细胞2h后,可获得最佳的转染效率(42.1%);G5PAMAM-D转染效率显著高于G2PAMAM-D(42.1%比19.4%,P<0.05);PAMAM-D基因转移系统对细胞的生长增殖无显著影响(P>0.05)。结论:PAMAM-D纳米载体在优化的转染条件下,可安全高效地介导目的基因转染Tca8113细胞,是一种较理想的基因转移系统。更多还原

【Abstract】 PURPOSE: Nanoparticle is an attractive and potential promising vector for cancer gene therapy. Transfection of cultured cells is also a common model system to test the effectiveness of nanoparticle-based vectors. The objective of this study was to optimize transfection conditions for polyamidoamine dendrimers (PAMAM-D) mediated enhanced green fluorescent protein gene (EGFP) in transfection of tongue carcinoma Tca8113 cells for obtaining ideal transfection efficiency and expression in vitro. METHODS: The expression and transfection efficiency of EGFP reporter gene in Tca8113 cells were explored by using PAMAM dendrimer- mediated gene transfer systems. A number of variables for maximal transfer of the DNA/dendrimer complexes were tested, including concentrations of DNA, weight ratios of DNA:dendrimer, dendrimer generations and transfection times. Comparison among the corresponding values of different groups was performed by one-way analysis of variance (one-way ANOVA), followed by student t test for duplicate comparisons. RESULTS: DNA/dendrimer complexes containing 1.0 μg of DNA and 2.0 μl of PAMAM-D (generation 5) incubated two hours resulted in highest gene expression (42.1%) in cultured cells. The transfection efficiency of generation 5 PAMAM-D was significantly higher than that of generation 2 PAMAM-D (42.1% vs 19.4%,P< 0.05). In addition, growth of the tumor cells was not influenced by PAMAM-D mediated gene transfer systems in all experiments(P>0.05). CONCLUSIONS: PAMAM dendrimers enable safe and highly efficient EGFP reporter expression in Tca8113 cells under optimized conditions. Furthermore, the data demonstrated that PAMAM dendrimer is an ideal gene transfer system for analysis of target gene expression in vitro.更多还原