核定位信号肽在超声靶向微泡破坏介导基因转染中的增强效应

摘要

Objective To investigate the transfection efficiency combining ultrasound targeted microbubbles destruction(UTMD)and nuclear localization signal(NLS)peptide for facilitating the plasmid of enhanced green fluorescent protein (pEGFP) into nucleus.Methods This study was divided into 3 groups,group A:UTMD+ pEGFP;group B:UTMD+ NLS + pEGFP;group C:Lipo3000 + pEGFP.The NLS was labeled by FITC and pEGFP was marked by Cy3.The different mole ratio was adjusted between NLS and pEGFP for observing the best ratio of combination.The human umbilical vein endothelial cells (HUVEC) were transfected by the optimum ultrasonic irradiation parameters and the optimal NLS∕pEGFP mole ratio.Six hours after transfection,the rate of Cy3 labeled pDNA into cells and nuclear were detected by flow cytometer and laser confocal microscope respectively.Forty-eight hours after transfection,the transfection efficiency was detected by flow cytometer;the survival rate of cells was measured by CCK8. RT-PCR and Western technology were used to detect the relative expression amount of mRNA and protein. The above indicators were compared among 3 groups,which were used to evaluate the enhanced effect of NLS in UTMD mediated gene transfection.Results ① Six hours after transfection,the NLS with green fluorescence and pEGFP with red fluorescence can show at the same site and signal intensity within the cell, that suggested a combination between them,agarose gel electrophoresis showed that the best molar ratio of NLS∕pEGFP combining was 10 4 ∶1 .②Six hours after transfection,the rates of pEGFP into the cells were (63±12)%,(80±10)% and(92±8)%;the rates of pEGFP into the nucleus were(1 7±3)%,(50±12)%and(35±8)% in 3 groups respectively(P <0.05).Compared with group A,both rates were 1 .3 and 2.9 times in group B and 1 .5 and 2.1 times in group C.③Forty-eight hours after transfection,the cell activity were>80% in all groups;the transfection efficiency,relative quantity of mRNA and protein expression were increased gradually.There were significant differences among 3 groups(P <0.05).They were 1 .6,2.3 and 2.4 times in group B than those in group A,still lower than those in group C.Conclusions The UTMD combining NLS can promote the pEGFP into nucleus for improving the transfection efficiency.The NLS peptide can play an enhanced effect as a new strategy of UTMD.%目的：利用超声靶向微泡破坏(UTMD)技术联合核定位信号(NLS)肽促进增强型绿色荧光蛋白质粒(pEGFP)入核,提高 UTMD 介导基因的转染效率。方法实验分3组：UTMD+pEGFP 组(A 组)、UTMD+ NLS 肽+ pEGFP 组(B 组)和阳离子脂质体 Lipo3000+ pEGFP 组(C组)。FITC 荧光标记 NLS,核酸染料 Cy3标记 pEGFP,调整 NLS 肽与 pEGFP 不同摩尔比,观测两者最佳结合比值。以最佳超声辐照参数及 NLS 肽/pEGFP 摩尔比转染人脐静脉内皮细胞(HUVEC)。转染6 h 后,用流式细胞仪和激光共聚焦显微镜分别评价 Cy3标记质粒的入胞率和入核率。转染48 h 后,用流式细胞仪检测转染效率,CCK8检测细胞存活率,RT-PCR 和 Western 分别检测 mRNA 和蛋白的相对表达量,并比较三组间的差异以评价 NLS 肽在 UTMD 介导基因转染中的增强效应。结果①转染6 h 后,带绿色荧光的 NLS 肽和红色荧光的 pEGFP 均能在细胞同一部位显示且强度信号一致,提示两者相互结合；琼脂糖凝胶电泳示最佳 NLS 肽/pEGFP 结合摩尔比为104∶1。②转染6 h 后,A、B、C 三组质粒的入胞率分别为(63±12)%、(80±10)%和(92±8)%,入核率分别为(17±3)%、(50±12)%和(35±8)%,差异均有统计学意义(P 均<0.05),B 组入胞率和入核率分别为 A 组的1.3倍和2.9倍, C 组分别为 A 组的1.5倍和2.1倍。③转染48 h 后,三组细胞活性均>80%,转染效率、mRNA 和蛋白表达的相对量均依次增加,差异均有统计学意义(P 均<0.05),B 组分别为 A 组1.6倍、2.3倍和2.4倍,但仍低于 C 组。结论 NLS 联合 UTMD 能提高 pEGFP 入核率,从而提高转染效率,NLS 肽发挥增强效应为 UTMD 介导转染提供新策略。