CAJ | 학술논문

目的:研究半乳糖化壳聚糖-低分子聚乙烯亚胺(galactosylated chitosan-graft-polyethyleneimine,GC-PEI)/DNA复合物在体内外的肝靶向性.方法:GC-PEI与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP) 质粒(pEGFP-Cl)在0.01 mol/L PBS,150 mmol/L NaCl,5%葡萄糖溶液中自组装成3种不同溶媒介导的GC-PEI/ DNA复合物,检测复合物粒径大小与形态,Zeta 电位以及结合和保护DNA 的能力;并进一步测定GC-PEI聚合物的毒性,研究复合物的肝靶向转染效率.结果:在GC-PEI与DNA质量比为1∶1～2.5∶1时,GC-PEI聚合物能有效地结合和保护所携带的DNA免受核酸酶和血清的降解.复合物粒子呈规则的球形,有明显的核壳结构.GC-PEI聚合物在检测细胞中未显示出明显毒性;动物体内急性毒性实验显示:通过尾静脉注射50～300 μg的GC-PEI聚合物入小鼠后,实验小鼠2周内无急性毒性反应和死亡发生.荧光显微镜和流式细胞仪检测证实GC-PEI/DNA复合物在肝细胞系(QSG7701/core,L02)中的绿色荧光蛋白(green fluorescent protein,GFP)表达明显高于非肝细胞系(SGC-7901,HBE)细胞.体内实验表明转染48 h后,小鼠肝组织在荧光显微镜下可以检测到明显的绿色荧光,而其他主要脏器未见明显荧光.结论:GC-PEI聚合物能够在体内外特异性将外源基因或DNA导入肝细胞,具有良好的肝靶向性.
목적:연구반유당화각취당-저분자취을희아알(galactosylated chitosan-graft-polyethyleneimine,GC-PEI)/DNA복합물재체내외적간파향성.방법:GC-PEI여증강형록색형광단백(enhanced green fluorescent protein,EGFP) 질립(pEGFP-Cl)재0.01 mol/L PBS,150 mmol/L NaCl,5%포도당용액중자조장성3충불동용매개도적GC-PEI/ DNA복합물,검측복합물립경대소여형태,Zeta 전위이급결합화보호DNA 적능력;병진일보측정GC-PEI취합물적독성,연구복합물적간파향전염효솔.결과:재GC-PEI여DNA질량비위1∶1～2.5∶1시,GC-PEI취합물능유효지결합화보호소휴대적DNA면수핵산매화혈청적강해.복합물입자정규칙적구형,유명현적핵각결구.GC-PEI취합물재검측세포중미현시출명현독성;동물체내급성독성실험현시:통과미정맥주사50～300 μg적GC-PEI취합물입소서후,실험소서2주내무급성독성반응화사망발생.형광현미경화류식세포의검측증실GC-PEI/DNA복합물재간세포계(QSG7701/core,L02)중적록색형광단백(green fluorescent protein,GFP)표체명현고우비간세포계(SGC-7901,HBE)세포.체내실험표명전염48 h후,소서간조직재형광현미경하가이검측도명현적록색형광,이기타주요장기미견명현형광.결론:GC-PEI취합물능구재체내외특이성장외원기인혹DNA도입간세포,구유량호적간파향성.
Objective To investigate the hepatocyte targeted specific property of galactosylated chitosan-graft-polyethyleneimine (GC-PEI)/DNA complexes in vitro and in vivo.Methods With the plasmid expressing enhanced green fluorescent protein (pEGFP-C1) as the reporter gene,the formation of GC-PEI/DNA complexes was induced to self-assemble in 0.01 mol/L phosphate buffered saline(PBS),150 mmol/L NaCl,or 5% glucose solution (GS).The complexes were characterized by the particle size,Zeta potential,DNA binding and protection capacity,and further tested for cytotoxicity and hepatocyte targeted degradation of DNaseⅠand the serum,which presented as a well-formed sphere or compacted nucleocapsid structure at a diameter of 50-200 nm.The GC-PEI copolymer showed no obvious toxicity in the tested cell lines.Acute toxicity assay revealed that the mice grew well in 2 weeks with GC-PEI dosage from 50 to 300 μg.The assay by flow cytometry and fluorescent microscope showed that the transfection efficiency in hepatocyte lines (L02,QSG7701/core) was higher than that in non-hepatocyte lines (SGC7901,HBE) in vitro.In vivo,the GFP was obviously expressed in the liver tissue and not expressed in other organs 48 h after the transfection.Conclusion GC-PEI copolymer may carry the exogenous gene specifically to hepatocytes in vitro and in vivo,which has very good liver targeted specific property.