【摘要】 背景与目的:选择合适的载体是基因治疗成功的关键之一,近年来一种新型多聚阳离子化合物——聚乙烯亚胺(polyethylenimine,PEI)作为高效低毒的基因转导载体得到广泛重视。本研究应用光化学法制备系列不同粒径的PEI纳米凝胶,初步探讨其转导效率与粒径之间的关系,筛选理想的肿瘤基因转导载体。方法:光化学法制备PEI,光相干光谱仪测定粒径,并用扫描电子显微镜和原子力显微镜表征;以PEI为载体将增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)报告基因分别转入Bel7402细胞和A549细胞,荧光显微镜下计数和流式细胞仪检测转染率。结果:光相干光谱仪检测PEI粒径38～200nm,扫描电子显微镜和原子力显微镜检测其形貌多为球形。荧光显微镜下计数和流式细胞仪检测86.9nmPEI(4μg)介导EGFP基因(2μg)时转染率最高,Bel7402细胞分别为(32.75±1.01)%、(32.40±1.41)%,A549细胞分别为(29.81±1.84)%、(30.00±1.86)%;与脂质体相比差异无统计学意义(P>0.05)。结论:光化学法合成的PEI是有效的基因转导载体,PEI粒径为86.9nm转染最为有效。更多还原

【Abstract】 BACKGROUND & OBJECTIVE: Choosing suitable gene carrier is very important in gene therapy. Recently,polyethylenimine (PEI),a new polycation compound,is particularly attractive due to its high transduction efficiency and low toxicity. This study was to synthesize a series of PEI nanogels of different particle diameters by photochemistry,investigate the relation between the transfection efficiency and particle diameter,and screen ideal gene carrier. METHODS: PEI nanogels were synthesized by photochemistry. The particle diameter was detected by photo correlation spectroscopy (PCS). The spherical morphology of the nanogels was characterized by scanning electron microscopy (SEM),and confirmed by atomic force microscopy (AFM). Using PEI/DNA complex as a plasmid vector,the enhanced green fluorescence protein (EGFP) gene was transferred into Bel7402 and A549 cells. Gene expression was quantitatively evaluated by fluorescent microscopy and flow cytometry (FCM). RESULTS: The diameter of synthesized PEI nanogels was in the range of 80-200 nm,and most of them were globular. The delivery rate reached the maximum when using 4 μg PEI (86.9 nm) to transfer 2 μg EGFP: the delivery rates were (32.75±1.01)% for Bel7402 cells and (29.81±1.84)% for A549 cells when detected by fluorescent microscopy,and were (32.40±1.41)% for Bel7402 cells and (30.00±1.86)% for A549 cell when detected by FCM. There was no significant difference between PEI and LipofectamineTM 2000 in the transfection efficiency (P > 0.05). CONCLUSIONS: PEI nanogels synthesized by photochemistry are effective nonviral vectors for gene delivery into human tumor cells in vitro. The transfection efficiency of 86.9 nm PEI is the highest.更多还原