新型非病毒载体聚乙烯亚胺介导基因转染参数的研究  

Gene Transfer by the New Type Nonviral Vector Polyethylenimine

作　　者：李经忠[1] 王青青[1] 余海[1] 

机构地区：[1]浙江大学免疫学研究所,杭州310031

出　　处：《中国生物医学工程学报》2006年第4期481-487,共7页Chinese Journal of Biomedical Engineering

基　　金：国家高技术研究发展计划资助项目(2001AA217071)

摘　　要：聚乙烯亚胺是一种新型阳离子多聚物基因释放载体,可结合浓缩DNA,通过粘附内吞,进入细胞,使携带的质粒表达。本研究目的通过对聚乙烯亚胺各种转染参数的测定,为合成以聚乙烯亚胺为骨架的人工载体积累数据。方法:本研究利用聚乙烯亚胺分别结合含β半乳糖甙酶报告基因的pSVβ表达质粒、含绿色荧光蛋白报告基因的pEGFP质粒转染Cos-7细胞,通过组织化学法测定细胞抽提产物中β半乳糖甙酶的表达量、流式细胞仪法测定绿色荧光蛋白阳性细胞的表达比例,来测定影响转基因效率的各种参数。结果:在培养液中,6μg/ml聚乙烯亚胺作用24h,NIH 3T3细胞生存率为64.2%,7μg/ml聚乙烯亚胺细胞生存率为54.4%。电泳阻滞试验,聚乙烯亚胺在N/P比在3.0以上方可完全结合DNA。溶酶体抑制剂氯喹可增加聚乙烯亚胺的转染效率。培养液中的白蛋白、血清可降低转染效率。作为配制聚乙烯亚胺/DNA复合物的溶媒,HEPES缓冲液优于生理盐水,生理盐水优于5%葡萄糖。配制聚乙烯亚胺/DNA复合物的溶媒中加入Mg2+降低转染效率。聚乙烯亚胺转染效率优于SuperFectTM(断裂型树突状多聚物),而毒性低于SuperFectTM。结论:本研究首次报道了聚乙烯亚胺与DNA结合配伍的N/P比计算公式,N/P=7.75×b/c,这里b是PEI的质量(μg),c是质粒的质量(μg);PEI的工作终浓度应≤6μg/ml。通过体外细胞试验证明,聚乙烯亚胺是一种有效的真核细胞转染剂和人工合成基因载体的骨架。Objective Polyethylenimine can condense DNA into granules of nanometer size for delivering gene into mammalian cells. The influencing factors on polyethylenimine （Mr 25000, branched） gene delivery efficiency were studied, so as to be used for in vivo gene therapy and synthesize more complicated nonviral gene delivery vector. Methods In vitro cytotoxic effects of polyethylenimine were quantified by MTT assay. The interaction between polyethylenimine and DNA at different charge ratios was analyzed by agarose gel electrophoresis retardation assay. The potential for transgene expression was monitored in Cos-7 cells using pEGFP and pSVβ plasmids. Influences of chloroquine, albumin, serum, salt ion strength, etc. on polyethylenimine/DNA transgene efficiency were assayed. Results The survival rate of NIH3T3 cells incubated with 6μg/ml of PEI and 7μg/ml of PEI for 24 hr was 64.2 % and 54.4%, respectively. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 polyethylenimine nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of polyethylenimine. Albumin,serum in the culture medium decreased the transfection efficiency. HBS（HEPES buffered solution） or 150mmol/L NaCl as the dilution solution of polyethylenimine/DNA excelled over 278mmol/L glucose solution in the transfection efficiency. The calculating equation of polyethylenimine nitrogens and DNA phosphates ratio （N/P ratio）, was first officially reported, namely, N/P ratio = 7.75×b/c, here ＂b＂ is the mass of PEI （μg）, ＂c＂ is the mass of plasmid DNA （μg）. Conclusion Polyethylenimine was efficient gene transfer agent of eukaryotes in vitro, and could be used for synthesizing more complicated gene delivery vectors.

关 键 词：聚乙烯亚胺 基因转移 合成载体 

分 类 号：R392-33[医药卫生—免疫学]