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RNA干扰技术在靶基因验证和人类肿瘤治疗研究方面都已显示出良好的应用前景.目前小干扰RNA(siRNA)体内应用较为理想的转运工具是非病毒载体,其中聚乙烯亚胺(PEI)以其高效性和通用性而最具代表性.本研究以一种线性化PEI为载体构建靶向表皮生长因子受体的siRNA转运系统,采用琼脂糖凝胶电泳阻滞实验,和以Lipofectamine 2000为对照的SPC-A1腺癌细胞转染实验,对决定LPEI/siRNA-EGFR复合物转染效率的N/P比条件进行优化,用Real time RT-PCR和流式细胞技术检测各组细胞的EGFR表达,最后以动态光散射技术和原子力显微镜对最优化条件下形成的LPEI/siRNA-EGFR复合物进行基本物理特性观察.结果显示,N/P≥5时siRNA可被LPEI充分聚合浓缩,且N/P=5时形成的LPEI/siRNA-EGFR复合物可引发最高效且特异的EGFR表达抑制,其效率与Lipofectamine 2000转染结果相当.该最优化N/P比条件下所形成的复合物是一种大小较为均一的椭圆形颗粒,其粒径小于100nm,表面带有较低的正电荷,该特性可有利于细胞摄取和内吞.以上研究表明,这种LPEI介导的以EGFR为靶向的siRNA转运系统极可能成为人肺腺癌基因治疗的新策略,对此仍需进一步的体内验证.
RNA interference technology has shown a good application prospect in the field of target gene validation and human cancer therapy research.Nowadays, the ideal transporter for small interfering RNA (siRNA) in vivo is a non-viral vector, in which polyethyleneimine (PEI) High efficiency and versatility and the most representative.In this study, a linearized PEI as a carrier targeting EGFR targeting siRNA transport system using agarose gel electrophoresis retardation experiments and Lipofectamine 2000 as a control The N / P ratio of LPEI / siRNA-EGFR transfection efficiency was optimized by transfection experiments of SPC-A1 adenocarcinoma cells. The expression of EGFR in each group of cells was detected by Real time RT-PCR and flow cytometry Finally, the basic physical properties of LPEI / siRNA-EGFR complexes formed under the optimal conditions were observed by dynamic light scattering and atomic force microscopy.The results showed that siRNA could be fully polymerized and condensed by LPEI when N / P≥5, and N / The formation of LPEI / siRNA-EGFR complex at P = 5 elicited the most efficient and specific inhibition of EGFR expression with comparable efficiency to that of Lipofectamine 2000. The complex formed under optimized N / P ratio was one Large species The more uniform oval particles, the particle size of less than 100nm, the surface with a lower positive charge, which is conducive to cellular uptake and endocytosis.The above studies have shown that this LPEI-mediated EGFR-targeted siRNA Transport system is likely to become a new strategy for gene therapy of human lung adenocarcinoma, which still needs further in vivo validation.