摘要
目的研究证实精氨酸修饰壳聚糖(ACS)制备的载基因纳米粒子显著地提高壳聚糖(CS)基因载体的转基因效率,该文进一步探索ACS摄入及跨膜机制对基因转染效率的影响。方法用荧光标记的CS或者ACS与荧光素酶质粒DNA复合制备CS载基因纳米粒子[壳聚糖载基因纳米粒子(CGN),精氨酸修饰壳聚糖载基因纳米粒子(ACGN)];与大鼠平滑肌细胞系A10细胞、不同跨膜通道的抑制剂(氯丙嗪、非律平和Dynasore)共孵育,以评价其基因递送的跨膜途径。通过与不同跨膜途径的抑制剂共转染,观察不同跨膜通道对转基因效率的影响。结果 ACGN细胞摄入量是CGN的2倍[(1.534±0.016)μg/mg蛋白质vs(0.755±0.007)μg/mg蛋白质]。与CGN相比,ACGN内吞通道有所改变,非律平(质量浓度5μg/mL,小窝蛋白内吞通道的特异抑制剂)能显著地抑制ACGN的细胞摄入(46.6%)和转基因效率(48.2%),而小窝蛋白内吞通道对CGN作用不明显。结论实验数据显示,ACS改变了基因载体的内吞通道,促进了基因纳米粒子的细胞摄入量,同时也大幅度地提高了转基因效率。
Objective To investigate the internalization mechanism of arginine--conjugated chitosan （ACS） gene nanoparticles （ACGN）. Methods Chitosan （CS） and ACS were labeled with Alexa Flour 488 and subsequently used for the preparation of fluorescent chitosan gene nanoparticles （FCGN） and fluorescent ACGN （FACGN）. The endocytic pathways by which the cells internalize ACGN or CGN were explored by incubating FCGN or FACGN with A10 cells in the presence of a variety of endocytic pathway inhibitors. The transfection efficiency of the nanopartieles incubated with these inhibitors were also investigated. Results Cellular uptake of ACGN was twice as high as that of CGN[（1.534 ±0.016） p,g/mg protein vs （0.755 ± 0.007） μg/mg protein]. The endocytic pathways involved in the internalization of ACGN were different from that of CGN. Filipin（5 μg/mg; a specific inhibitor of the caveolin -mediated endocytosis） notably decreased the cellular uptake （46.6 %） and transgenic efficacy （48.2 %） of ACGN. However, inhibition of caveolin-mediated endocytosis had no significant effect on the internalization of CGN. Conclusion It is demonstrated that conjugation of arginine onto CS molecules enhanced the cellular uptake of ACGN and their transgenic efficacy through changing the endocytic pathways.
出处
《生物医学工程与临床》
CAS
2013年第2期172-177,共6页
Biomedical Engineering and Clinical Medicine
基金
科技部重大科学研究计划(2006CB933203)
天津市重点基金(09JCZDJC18700
11JCZDJC20300)
