壳糖体外介导血管平滑肌细胞内皮型一氧化氮合酶基因转移的初步研究

目的探讨壳糖作为非病毒载体介导质粒体外转染哺乳动物平滑肌细胞的可行性、转染效率及影响转染效率的因素。方法用壳糖包被携带绿色荧光蛋白(GFP)基因和内皮型一氧化氮合酶(eNOS)基因的质粒PAdTrack-CMV-eNOS,制备壳糖/质粒复合体(CPC-eNOS),在不同条件下将其和Wistar大鼠血管平滑肌细胞孵育,通过检测靶细胞是否表达GFP和eNOS,评价壳糖介导质粒转染的效率。并以裸质粒PAdTrack-CMV- eNOS和重组腺病毒表达载体Ad-eNOS分别作为对照。结果荧光显微镜下472nm观察,CPC-eNOS和重组腺病毒表达载体Ad-eNOS孵育的血管平滑肌细胞均观察到绿色荧光,Western印迹法检测均有eNOS蛋白表达,但裸质粒PAdTrack-CMV-eNOS组均为阴性。CPC-eNOS中氨基/磷酸基比值(N/P比值)为5、pH值为7.0时,对平滑肌细胞的转染效率最高。结论CPC-eNOS能够体外介导转染哺乳动物的血管平滑肌细胞。转染效率与CPC-eNOS的N/P比值和转染介质pH值有关。

Chitosanin/plasmid complex-mediated eNOS gene transfer into rat vascular smooth muscle cells in vitro

Objective To study the feasibility of chitosanin/plasmid complex-mediated transfection of eNOS in to vascular smooth muscle cells(VSMCs)of wistar rats.Methods PAdTrack-CMV-eNOS harboring green fluorescent protein(GFP)was coated with chitosanin to prepare chitosan/plasmid complexes(CPC);CPC were incubated with cultured VSMCs of Wistar rats aorta under different conditions.The transfection efficiency of CPC was evaluated by detecting GFP and eNOS in the target cells.Transfection with naked PAdTrack-CMV-eNOS plasmid and recombinant Ad-eNOS served as control.Results Evident fluorescence was observed in VSMCs transfected by CPC(highest expression was found at N/P ratio 5:1 at medium pH 7.0)and by Ad-eNOS; Western blotting also showed expression of eNOS protein;no fluorescence was observed in naked PAdTrack-CMV- eNOS transfected group.CPC-eNOS had the highest transfection efficiency of smooth muscle cells when the nitrogen:phosphorus ratio was 5 and the pH value was 7.Conclusion CPC can efficiently mediate gene transfection into VSMCs of Wistar rats and the transfection efficiency is related to the N/P ratio of CPC and the pH value of the medium.