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目的:利用阳离子多聚物多聚乙烯亚胺(PEI)纳米凝胶将增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)转入同步化的肿瘤细胞中并检测绿色荧光蛋白的表达率,探讨PEI作为载体介导肿瘤细胞基因转染中细胞周期对转染效率的影响。方法:通过6 MV X线照射及药物阻滞使Hela及A549细胞同步化,在5个PEI/DNA梯度比(2/2、4/2、6/2、8/2、10/2μg/μg)下利用PEI介导EGFP报告基因转染进入上述同步化细胞,流式细胞仪和荧光显微镜下测定绿色荧光蛋白表达率,通过梯度试验分析细胞周期时相与表达率之间的关系。结果:在不同增殖状态及PEI/DNA比值梯度下,一定剂量-时间的X射线和顺铂可以使Hela细胞同步化于S期,一定剂量-时间的艾素可以使A549细胞同步化于G2/M期,而血清饥饿法培养则可以使细胞阻滞于G0/G1期,在不同细胞周期时相下EGFP的转染效率不同,两种同步化细胞均表现出S期及G2/M期细胞的转染率显著高于对照组(P<0.05)和G0/G1期细胞(P<0.0001)。结论:PEI导入基因的表达效率受细胞周期制约,PEI介导的基因转染受到宿主细胞摄取、转运入核及转录能力的影响,而该进程都是细胞周期依赖性的。
OBJECTIVE: To transfer enhanced green fluorescence protein (EGFP) into synchronized tumor cells by cationic polymer polyethylenimine (PEI) nanoegel and to detect the expression of green fluorescent protein PEI as a vector to mediate the effect of cell cycle on the transfection efficiency in tumor cell gene transfection. METHODS: Hela cells and A549 cells were synchronized by 6 MV X-ray irradiation and drug blockade. Five PEI / DNA gradient ratios (2 / 2,4 / 2,6 / 2,8 / 2 and 10 / 2μg / ), PEI-mediated EGFP reporter gene transfection into the synchronized cells, flow cytometry and fluorescence microscopy to determine the expression of green fluorescent protein, gradient analysis of the relationship between the cell cycle phase and the expression rate. Results: Under certain gradient of proliferation and PEI / DNA ratio, a certain dose-time of X-ray and cisplatin could synchronize Hela cells in S phase. A dose-time of Alzheimer’s could synchronize A549 cells to G2 / M phase, and serum starvation culture can make cells arrested in the G0 / G1 phase, the transfection efficiency of EGFP in different cell cycle phases are different, both synchronized cells showed S phase and G2 / M phase cells Transfection rate was significantly higher than the control group (P <0.05) and G0 / G1 phase cells (P <0.0001). CONCLUSION: The expression of PEI-introduced genes is regulated by the cell cycle. The PEI-mediated gene transfection is affected by the uptake and transport of host cells into the nucleus and the transcriptional capacity. All these processes are cell cycle-dependent.