超声靶向微泡破坏联合经典核定位信号肽促进基因转染的实验研究

摘要

Objective To investigate the transfection efficiency of combining ultrasound targeted microbubble destruction (UTMD) and classic nuclear localization signal (cNLS) peptide for facilitating targeted gene into nucleus.Methods The plasmid of enhanced green fluorescent protein (pEGFP)was transfected into the 293T cells by different ways.This study was divided into four groups:control group, UTMD+pEGFP;cNLS group,UTMD+ pEGFP + cNLS;mutative group,UTMD+ pEGFP + mNLS;and retardant WGA group,UTMD+ pEGFP+ cNLS+ WGA.The NLS was labeled by FITC and pEGFP was maked by Cy3.Six hours after transfection,the rate of plasmid into cells was detected as the percentage of Cy3 positive cells by flow cytometry,the rate of plasmid into nuclear was calculated as the percentage of Cy3 fluorescence intensity in nucleus to the whole cell by laser confocal microscope.At 48 after transfection,the transfection efficiency was detected by flow cytometry,the survival rate of cells was measured by CCK8. RT-PCR and Western blot were used to detect the relative expression amount of mRNA and protein.The differences were compared in four groups to evaluate the enhancement effects of cNLS during UTMD mediated gene transfection.Results ①At 6 hours after transfection,almost all green fluorescence showed in the nucleus in cNLS group,it appeared in both the cytoplasm and nucleus in mNLS group,but mostly appeared in the cytoplasm and almost none in nucleus in WGA group.②At 6 hours after transfection,the rate of pEGFP into the cells were(61 ±1 1)%,(80±10)%,(55±9)% and(58±10)%;the rate of pEGFP into the nucleus were(20±4)%,(50±1 1)%,(1 8±3)% and (10±3)% in four groups respectively.The rates were highest in cNLS group and lowest in WGA group,the difference was statistically significant(P <0.05).③ At 48 hours after transfection,the cell activity were > 80% in all groups;the transfection efficiency,the relative quantity of mRNA and protein were highest in cNLS group and lowest in WGA group,the difference was statistically significant(P <0.05).Conclusions The UTMD combining cNLS can promote pEGFP into the nucleus for improving the transfection efficiency.%目的：利用超声靶向微泡破坏(UTMD)技术联合经典核定位信号肽(cNLS)促进目的基因入核,提高转染效率。方法用不同的方式将增强型绿色荧光蛋白质粒(pEGFP)转入293T 细胞。实验分4组：UTMD+pEGFP (对照组),UTMD+pEGFP+cNLS (经典型组),UTMD+pEGFP+mNLS (突变型组)和 UTMD+pEGFP+cNLS+WGA (入核阻滞组)。NLS 和 pEGFP 分别用 FITC 和Cy3标记。转染6 h 后,流式细胞仪检测 Cy3阳性细胞百分比作为质粒入胞率,激光共聚焦显微镜观测细胞核中 Cy3荧光强度占整个细胞荧光强度百分比作为质粒的入核率。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 blot 分别检测 mRNA 和蛋白的相对表达量,并比较上述指标在4组间的差异以评价 cNLS 在 UTMD 介导基因转染中的作用。结果①转染6 h 后,带绿色荧光标记的 NLS 在细胞内不同部位显示,经典型组大量入核,突变组细胞质和细胞核均可显示,入核阻滞组主要聚集在细胞质,未能入核；②转染6 h 后,4组质粒的入胞率分别为(61±11)%、(80±10)%、(55±9)%和(58±10)%；入核率分别为(20±4)%、(50±11)%、(18±3)%和(10±3)%,经典组入核率和入胞率最高,入核阻滞组最低,差异有统计学意义(P <0.05)。③转染48 h后,4组细胞活性均>80%；转染效率、mRNA 和蛋白相对表达量均在经典组最高,入核阻滞组最低,差异均有统计学意义(P <0.05)。结论 UTMD 联合 cNLS 能提高 pEGFP 入核率,从而提高目的基因转染效率。