Okazaki Arimichi, Jo Jun-Ichiro, Tabata Yasuhiko
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Tissue Eng. 2007 Feb;13(2):245-51. doi: 10.1089/ten.2006.0185.
A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.
设计了一种新的非病毒基因转染方法，以提高大鼠间充质干细胞（MSCs）的基因表达水平。通过化学引入精胺对支链淀粉进行阳离子化处理，制备非病毒载体阳离子化支链淀粉（精胺-支链淀粉）。将精胺-支链淀粉与荧光素酶的质粒脱氧核糖核酸（DNA）复合，并与人工细胞粘附蛋白的前胶原蛋白一起包被在培养底物表面。将MSCs在复合包被的底物上进行培养和转染（反向转染），并将基因表达水平和持续时间与在含有质粒DNA-精胺-支链淀粉复合物的培养基中培养转染的MSCs（传统方法）进行比较。与传统方法相比，反向转染方法显著增强并延长了基因表达。与传统方法不同，反向方法允许在有血清的情况下对MSCs进行转染培养，传统方法为细胞提供了良好的培养条件以降低细胞毒性。使用搅拌和振荡培养方法对涂覆有复合物和前胶原蛋白的聚对苯二甲酸乙二酯（PET）无纺布进行反向转染。这两种方法比静态方法显著增强了MSCs的基因表达水平和持续时间。介质循环可能在氧气和营养供应以及废物排泄方面改善细胞的培养条件，从而导致基因表达增强。
