壳寡糖纳米载体细胞内递送EGFR脱氧核酶研究.docVIP

壳寡糖纳米载体细胞内递送EGFR脱氧核酶研究 　　摘 要：设计靶向EGFR mRNA的脱氧核酶(EGFR DRz)，以壳寡糖(COS)为材料，建立了一种有效的纳米基因细胞内传递体系，并研究其介导的靶向EGFR的脱氧核酶在Hela细胞内的生物学效应.流式结果表明COSEGFR DRz复合体转染效率为88.7%，与脂质体转染试剂的89.7%相比无显著差异.半定量RTPCR结果显示，经壳寡糖纳米载体递送的EGFR DRz能有效地靶向切割Hela细胞内的EGFR mRNA，使其表达下降.进一步的流式分析显示细胞被阻滞在G0~G1期，并且出现凋亡现象，其中COS组的凋亡率为??19.3%，??大于对照组脂质体的凋亡率13.0%.研究表明，COS较脂质体有相似的转染效率和更低的毒性，是一种潜在的、有效的脱氧核酶递送载体. 　　关键词：壳寡糖；脱氧核酶；EGFR；基因递送 　　中图分类号：Q74 文献标识码：A 　　 　　Experimental Study of the Oligochitosan Nanoparticles 　　of Intracellular EGFR DNAzyme Delivery 　　LI Dan,WANG Bei, LIN Yi??ting, JIN Ran, LIU Zhi??wen, LIU Xuan??ming???k 　　(Research Center of Biological Energy Sources and Biomaterial, School of Biology, 　　 Hunan Univ, Changsha, Hunan 410082, China) 　　Abstract: 　　 A new intracellular gene delivery system based on oligochitosan (COS) was prepared and proven to be effective. First of all, The DNAzyme targeting EGFR (EGFR DRz) was designed and synthesized. Then, the biological effect of the EGFR DRz delivery system based on COS was studied in Hela cell with Flow Cytometry. It was shown that there was no statistically significant difference in transfection efficiency between liposome transfection reagent (89.7%)and COS??EGFR DRz (88.7%) . The results of semiquantitative RT??PCR confirmed that the EGFR mRNA in Hela cell could be cleaved by targeting COS??EGFR DRz. The cell cycle analysis in Hela cells transfected with COS??EGFR DRz revealed an arrestment in the G0/G1 phase. And the cell apoptosis rate induced by COS??EGFR DRz was 19.3%, which was higher than the rate induced by liposome??EGFR DRz (13.0%). These results have demonstrated that COS possesses higher transfection efficiency and lower cytotoxicity. It will be a potential DNAzyme delivery system. 　　Key words:oligochitosan; DNAzyme; EGFR; gene delivery 　　 　　脱氧核酶(DNAzyme， DRz)是一种能特异性结合并切割靶RNA分子的酶性DNA，具有高效的催化降解能力和反义靶向识别能力［1］，被广泛用于肿瘤、遗传病等疾病的研究.脱氧核酶作为一种新型的靶基因沉默手段，其最主要的问题之一是如何有效地进入靶细胞.因此，寻找一种相容性好、在生物体内能够稳定递送基因的材料在基因治疗中具有广泛应用的价值和意义. 　　作为壳聚糖的降解产物，壳寡糖(o