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。
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.Thenβ-galactosidase(β-Gal)in situ staining assay,Western blot assay and Caspase-3 activity assay are executed.The results show thatβ-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 andβ-Gal co-delivery group.All these results provide a reliable experiment basis for further application of PEI in gene and protein co-delivery.
作者
刘艳红
李静
王娜
李坤
张骥
余孝其
LIU Yanhong;LI Jing;WANG Na;LI Kun;ZHANG Ji;YU Xiaoqi(Experiment Teaching Center of Basic Chemistry,Sichuan University,Chengdu 610064,China;College of Chemistry,Sichuan University,Chengdu 610064,China)
出处
《实验室研究与探索》
CAS
北大核心
2022年第9期45-49,共5页
Research and Exploration In Laboratory
基金
国家自然科学基金项目(21877082)
四川大学教改项目(SCU9132)
四川大学研究生院教改项目(GSSCU2021093)。
