中国临床药理学与治疗学2011，Vol.16Issue(11)：1227-1233,7.
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
摘要
Abstract
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 up-regulation of p53 mRNA and p53/p21/Rb protein expression levels, and the obvious downreg-ulation 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.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 up-regulation of p53 mRNA and p53/p21/Rb protein expression levels, and the obvious downreg-ulation 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.关键词
尿刊酸修饰的壳聚糖/野生型p53/基因转染/人肝癌细胞系/细胞周期阻滞Key words
Urocanic acid-modified chitosan/ Wild-type p53/ Gene transfection/ Human hepatoma cell line/ Cell cycle arrest分类
医药卫生引用本文复制引用
王伟,王玉,王若宁,徐苏颖,丁杨,周建平..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[J].中国临床药理学与治疗学,2011,16(11):1227-1233,7.基金项目
江苏省自然科学基金资助项目(BK2011624) （BK2011624）
江苏省高校长自然科学研究项目(10kjb310007) （10kjb310007）
中央高校基本科研业务费专项资金项目(JKQ2009017) （JKQ2009017）
省级大学生实践创新训练计划项目 （）
