肝细胞靶向性Gal-Bu对大鼠肝细胞的转染效率检测  

Detection of transfection efficiency of hepatocyte-targeting Gal-Bu in rat liver cells

作　　者：王玉强[1] 盛净[2] 陈书艳[1] 苏靖[3] 

机构地区：[1]上海交通大学医学院附属新华医院老年科,上海200092 [2]上海交通大学医学院附属第九人民医院老年科,上海200011 [3]上海交通大学药学院,上海200240

出　　处：《上海交通大学学报（医学版）》2013年第6期759-762,共4页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81001416;30973152;81270205);上海市科委基金(10JC1408902)~~

摘　　要：目的检测肝细胞靶向性半乳糖基化聚乙烯亚胺衍生物Gal-Bu对大鼠肝细胞(BRL-3A)的转染效率。方法对PEI-Bu化学修饰以半乳糖基团制备Gal-Bu,琼脂糖凝胶电泳检测其复合质粒DNA的能力;MTT法检测Gal-Bu对BRL-3A的细胞毒性;以荧光素酶质粒作为报告基因,测定Gal-Bu在BRL-3A细胞的转染效率;半乳糖竞争抑制实验观察Gal-Bu对肝细胞的靶向性。结果琼脂糖凝胶电泳表明在质量比>15时,Gal-Bu具有复合DNA的能力。在5～100μg/mL浓度范围内,Gal-Bu和PEI 25 000的细胞毒性随浓度升高而增大;在同一浓度下,Gal-Bu的细胞毒性小于PEI 25 000(P<0.01)。Gal-Bu/DNA在质量比为50时达到最高转染活性,其数值为PEI 25 000的5.6倍(P<0.01),且接近Lipofectamine 2000。半乳糖竞争抑制实验表明,100 mmol/L的半乳糖可显著降低Gal-Bu的转染效率(P<0.01),但是并不影响PEI-Bu的转染效率(P>0.05)。结论 Gal-Bu是一种低细胞毒性、高转染活性的非病毒肝细胞靶向基因载体,在肝脏疾病的基因治疗领域中具有潜在的应用前景。Objective To evaluate the transfection efficiency of hepatocyte-targeting galactosylated polyethylenimine derivative Gal-Bu in rat liver cells （BRL-3A）. Methods Gal-Bu was synthesized through chemical modification of PEI- Bu with galactose residue. The pDNA condensation ability of the polymer was evaluated by agarose gel electrophoresis, MTT assay was employed to detect the cytotoxicity of the polymer in BRL-3A cells, luciferase plasmid was used as the reporter gene to determine the transfection efficiency of Gal-Bu in BRL-3A ceils, and competition assay of galactose was performed to investigate the hepatoeyte-targeting property of Gal-Bu. Results Gel retardation assay showed complete condensation of pDNA at weight ratio 〉 15. At concentrations ranging from 5 to 100 ixg/mL, cytotoxicity of Gal-Bu and PEI 25 000 increased with the concentrations. Ga｜-Bu exhibited lower cytotoxicity than PEI 25 000 at the same concentration （P 〈 0.01）. The polymer performed the highest transfection efficiency at weight ratio of 50, which was 5.6 times of PEI 25 000 （P 〈 0.01） and was close to Lipofectamine 2000. Competition assay of galactose revealed that the transfection efficiency of Gal-Bu was significantly decreased in the presence of 100 mmol/L galactose （P 〈 0.01）, whereas this phenomenon was not observed on the transfection efficiency of PEI-Bu （P 〉 0.05）. Conclusion Gal-Bu is a non-viral hepatocyte-targeting gene carrier with lower cytotoxicity and enhanced transfection efficiency, which would be a promising candidate in hepatocyte-targeting gene therapy.

关 键 词：肝细胞靶向 非病毒基因载体 半乳糖 转染效率 细胞毒性 

分 类 号：R341[医药卫生—基础医学] Q789[生物学—分子生物学]