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目的:构建以香菇多糖为骨架材料键合低分子量聚乙烯亚胺的新型转基因载体,研究其在肿瘤细胞中的基因转染。方法:从香菇中提取香菇多糖作为载体骨架,1,1’-二羰基咪唑(CDI)作为交联剂,键合低分子量的聚乙烯亚胺(PEI1.2kDa)形成香菇多糖-聚乙烯亚胺(LNT-PEI)聚合载体材料。将叶酸(folicacid,FA)偶联到LNT-PEI上,形成具有肿瘤细胞靶向性的组装式基因载体LNT-PEI-FA。用核磁共振氢谱(1H-NMR)、傅里叶转换红外光谱(FT-IR)和热重分析(TGA)等对载体材料进行化学表征,以确定其结构。用凝胶电泳阻滞实验观察LNT-PEI-FA对质粒DNA的结合能力,MTT法检测聚合物的毒性,用TEM对LNT-PEI-FA/DNA复合物的粒径大小、形状等进行检测。在A293和B16细胞株上进行转染实验。结果:凝胶阻滞电泳结果显示,LNT-PEI-FA与DNA在W/W为1.8∶1时可以完全缩合DNA,在相同浓度下LNT-PEI-FA的毒性明显低于PEI25kDa;体外转染实验显示,在A293和B16细胞株上具有很高的转染效率,荧光素酶表达值分别为1×1010和1×107。结论:以香菇多糖为骨架材料键合低分子量聚乙烯亚胺形成的转基因载体是一种有潜在研究价值的新型非病毒转基因载体。
OBJECTIVE: To construct a new type of transgene vector with low molecular weight polyethylene imine bound to lentinan as matrix material and study its gene transfection in tumor cells. Methods: Lentinan was extracted from shiitake mushrooms as carrier, and 1,1’-dicarbonylimidazole (CDI) was used as a cross-linking agent to bond low molecular weight polyethyleneimine (PEI1.2kDa) to form lentinan-polyethyleneimine (LNT-PEI) polymeric carrier material. Folic acid (FA) was coupled to LNT-PEI to form LNT-PEI-FA, an assembled gene carrier targeting tumor cells. The structure of the support material was characterized by 1H-NMR, FT-IR and TGA. The binding ability of LNT-PEI-FA to plasmid DNA was observed by gel electrophoresis. The toxicity of polymer was detected by MTT assay. The particle size, shape and the like of LNT-PEI-FA / DNA complex were detected by TEM. Transfection experiments were performed on A293 and B16 cell lines. Results: Gel electrophoresis showed that LNT-PEI-FA and DNA could completely condense DNA at a W / W ratio of 1.8: 1. The toxicity of LNT-PEI-FA at the same concentration was significantly lower than that of PEI at 25kDa. Experiments show that A293 and B16 cell lines with high transfection efficiency, luciferase expression values were 1 × 1010 and 1 × 107. Conclusion: The transgene vector with low molecular weight polyethylenimine bonded with lentinan as a framework material is a novel non-viral gene delivery vector with potential research value.