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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 study the effect of genetically modified nanoparticles loaded with arginine-modified chitosan (ACS) on the transgene efficiency of chitosan (CS) gene carrier. This study further explored the effect of ACS on the gene transfection efficiency Impact. Methods CS-loaded gene nanoparticles [chitosan-loaded gene nanoparticles (CGN) and arginine-modified chitosan-loaded gene nanoparticles (ACGN)] were prepared by fluorescence-labeled CS or ACS combined with luciferase plasmid DNA. Rat smooth muscle cell line A10 cells were co-incubated with inhibitors of different transmembrane pathways (chlorpromazine, fenofibrate and Dynasore) to evaluate their transmembrane pathways for gene delivery. The effect of different transmembrane channels on transgene efficiency was investigated by co-transfection with inhibitors of different transmembrane pathways. Results The amount of ACGN cells was twice that of CGN [(1.534 ± 0.016) μg / mg protein vs (0.755 ± 0.007) μg / mg protein]. Compared with CGN, the ACGN endocytosis pathway was changed. The phenylalanine (5μg / mL concentration-specific inhibitor of caveolin endocytosis) could significantly inhibit the cellular uptake of ACGN (46.6%) and the transgene efficiency (48.2%), while the caveolin protein endocytosis has no obvious effect on CGN. Conclusions Experimental data showed that ACS changed the endocytosis pathway of gene carriers, promoted the cell uptake of gene nanoparticles and greatly improved the transgene efficiency.