上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2013, Vol. 33 ›› Issue (6): 759-.doi: 10.3969/j.issn.1674-8115.2013.06.012

• 论著（基础研究） • 上一篇    下一篇

肝细胞靶向性Gal-Bu对大鼠肝细胞的转染效率检测

王玉强1，盛 净2，陈书艳1，苏 靖3   

  1. 上海交通大学 1.医学院附属新华医院老年科， 上海 200092； 2.医学院附属第九人民医院老年科， 上海 200011； 3.药学院， 上海 200240
  • 出版日期:2013-06-28 发布日期:2013-06-28
  • 通讯作者: 陈书艳, 电子信箱: shuyanchencn@yahoo.com.cn; 苏靖, 电子信箱: jingsu@sjtu.edu.cn。
  • 作者简介:王玉强（1985—）， 男， 博士生; 电子信箱: wangyuqiangabc@163.com。
  • 基金资助:

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

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

WANG Yu-qiang1, SHENG Jing2, CHEN Shu-yan1, SU Jing3   

  1. 1.Department of Geriatrics, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China; 2.Department of Geriatrics, the Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China; 3.School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China
  • Online:2013-06-28 Published:2013-06-28
  • Supported by:

    National Natural Science Foundation of China, 81001416, 30973152, 81270205; Shanghai Science and Technology Committee Foundation, 10JC1408902

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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5~100 μg/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。

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

Abstract:

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 cells, and competition assay of galactose was performed to investigate the hepatocyte-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 μg/mL, cytotoxicity of Gal-Bu and PEI 25 000 increased with the concentrations. Gal-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.

Key words: hepatocyte-targeting, non-viral gene carrier, galactose, transfection efficiency, cytotoxicity