新型阳离子基因传递载体PEI-PBLG的细胞转染研究  

Transfection with a Novel Cationic Gene Carrier: PEI-PBLG

作　　者：尉继征[1] 林磊[1] 熊伟[1] 祝庆余[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物工程学报》2007年第2期229-234,共6页Chinese Journal of Biotechnology

基　　金：北京市自然科学基金(No.7042051);高分子物理与化学国家重点实验室科学项目基金资助~~

摘　　要：对新型阳离子聚合物PEI（10kD）-PBLG进行研究，重点考察其基因转染效率与细胞毒性，探讨其作为基因载体的可能性。通过粒径分析及扫描电镜（SEM）观察PEI（10kD）-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径，预测其进入细胞的可能性。使用MTT比色法分析PEI（10kD）-PBLG、PEI（25kD）．PBLG、PEI（10kD）和PEI（25kD）的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型，将其与PEI（10kD）-PBLG自组装后，分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304，应用流式细胞术检测细胞转染效率，并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI（10kD）-PBLG可包裹质粒形成粒径100—120nm的纳米复合物，适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低，并能够在10％血清存在的条件下，转染全部实验用细胞株，尤其对Hela的转染效率最高，其次是COS-7、Vero-E6和ECV304；其中PEI-PBLG（10kD）／pEGFP复合物转染Hela细胞的比率为45．02％，高于Pri（10kD）／pEGFP的29．16％；PEI（10kD）-PBLG的细胞毒性作用显著低于PEI（25kD）、PEI（10kD）和PEI（25kD）-PBLG。新型阳离子多聚物PEI（10kD）-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性，提高了生物相容性，有望成为基因转移的有效载体。This study mainly deals with cell transfection and cytotoxicity for PEI（ 10kD）-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI （10kD）-PBLG was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） colorimetric assay and compared with PEI（25kD）-PBLG, PEI（10kD） , and PEI（25kD） . Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI （10kD）-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI（10kD） -PBLG/pDNA complex was approximately 100 - 200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI （10kD）-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI （ 10kD）-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression （45.02%） in Hela cells, which was considerably higher than that of PEI（ 10kD）/pEGFP （29.16%）. Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.

关 键 词：聚乙烯亚胺 聚谷氨酸 基因传递 转染效率 细胞毒性 

分 类 号：R346[医药卫生—基础医学]