新型阳离子聚合物非病毒载体介导野生型p53基因体外转染效果的研究

Study on effects of novel cationic polymer nonviral vector-mediated wild-type p53 gene transfection in vitro

目的:研究新型阳离子聚合物非病毒载体———尿刊酸修饰的壳聚糖(UAC)介导野生型p53(wt-p53)基因体外转染人肝癌细胞系HepG2的效果。方法:载体UAC包裹含绿色荧光蛋白(GFP)报告基因和wt-p53治疗基因的质粒(pEGFP-p53)转染HepG2细胞,利用流式细胞术和荧光显微术检测细胞内GFP的表达,优选最佳转染条件;采用RT-PCR法检测细胞内p53mRNA的表达,流式细胞术评价基因转染对细胞周期的影响,Western blot法检测细胞内p53及其下游细胞周期调控蛋白的表达。结果:在低pH(pH=6.9)条件下,采用低壳聚糖分子量(20000)的UAC作为载体,与pEGFP-p53形成高N/P(N/P=30)的UAC/pEGFP-p53复合物转染HepG2细胞,可获得最佳转染效果;载体UAC介导wt-p53基因转染细胞后,p53mRNA表达水平明显上调,G0/G1期出现显著阻滞,p53、p21和Rb蛋白表达水平明显上调,cyclin D1和CDK4蛋白表达水平明显下调。结论:载体UAC可成功介导wt-p53基因转染HepG2细胞,并诱导其产生细胞周期阻滞。

AIM：To investigate the effects of novel cationic polymer nonviral vector urocanic acid-modified chitosan（UAC）-mediated wild-type p53（wt-p53） gene transfection in human hepatoma cell line HepG2 in vitro.METHODS： UAC packaged the plasmid pIRES2-EGFP-p53（pEGFP-p53） to form the UAC/pEGFP-p53 complexes which transfected HepG2 cells,and the pEGFP-p53 contained a report gene of green fluorescent protein（GFP） and a therapeutic gene of wt-p53.The GFP expression in cells was assayed by flow cytometry and fluorescence microscopy to screen the optimum transfection conditions.The expression of p53 mRNA in cells was analyzed by RT-PCR,cell cycle arrest was assessed by flow cytometry,and the expression of p53 and its downstream cell cycle regulatory proteins was assayed by Western blot.RESULTS：Under the conditions of the low pH（pH=6.9）,high N/P（N/P=30）,and low chitosan molecular weight（20000）,the optimum transfection effect of UAC/pEGFP-p53 complexes in HepG2 cells could be obtained;UAC-mediated wt-p53 gene transfection in cells resulted in the significant G0/G1 phase arrest,the obvious upregulation of p53 mRNA and p53/p21/Rb protein expression levels,and the obvious downregulation of cyclin D1 and cyclin-dependent kinase（CDK） 4 protein expression levels.CONCLUSION： Wt-p53 gene could be successfully transfected into HepG2 cells mediated by UAC,which induced cell cycle arrest of HepG2 cells.