【摘要】 通过构建载体/基因/蛋白共递送复合体系，探讨了阳离子聚合物聚乙烯亚胺(PEI,25 kDa)负载基因和蛋白在细胞中共递送的效率。实验采用凝胶阻滞电泳确定了聚乙烯亚胺与蛋白、基因形成稳定复合物的最佳质量比为2∶1∶0.05。激光扫描共聚焦成像结果表明，聚乙烯亚胺可以有效递送Cy5-pDNA和FITC-BSA进入细胞。通过β-半乳糖苷酶(β-Gal)原位染色法、Western blot和Caspase-3活性检测实验，进一步分析了递送蛋白β-Gal的活性和p53基因的转录翻译功能。结果显示，PEI介导递送的β-Gal保持了较高的酶活性，可以有效催化底物的分解。共负载p53基因转染细胞后，p53蛋白表达水平显著上调且可以激活p53蛋白相关信号通路中关键性激酶Caspase-3。更多还原

【Abstract】 To investigate the dual delivery efficiency of gene and protein mediated by polyethyleneimine(PEI, 25 kDa), a co-delivery complexes system of PEI/gene/protein is constructed. The optimal mass ratio of PEI/protein/gene is 2∶1∶0.05 which is determined using gel retardation electrophoresis assay. Confocal laser scanning imaging results show that PEI/Cy5-pDNA/FITC-BSA complexes are effectively delivered into cells. To confirm the activity of protein and gene loaded by PEI, β-Gal gene and p53 gene are dual carried into A549 cells. Then β-galactosidase(β-Gal) in situ staining assay, Western blot assay and Caspase-3 activity assay are executed. The results show that β-Gal protein maintains high activity which can effectively catalyze the substrates. Furthermore, protein level of p53 is assessed. In PEI/p53/β-Gal complexes co-delivery group, P53 expression remarkable increases. Meanwhile, by analyzing caspase-3 activity, a key kinase in p53-related signaling pathway, shows significant enhancement in p53 and β-Gal co-delivery group. All these results provide a reliable experiment basis for further application of PEI in gene and protein co-delivery.更多还原