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

中国临床药理学与治疗学 ›› 2011, Vol. 16 ›› Issue (11): 1227-1233.

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

王伟¹, 王玉², 王若宁¹, 徐苏颖¹, 丁杨¹, 周建平¹

¹中国药科大学药剂学教研室,天然药物活性物质与功能国家重点实验室,南京 210009;
²南京医科大学药理学系,南京 210029, 江苏

收稿日期:2011-10-10 修回日期:2011-11-07 出版日期:2011-11-26 发布日期:2011-11-29
通讯作者: 周建平,男,博士,教授,博导,研究方向:肿瘤基因治疗。Tel: 025-83271272 E-mail: zhoujpcpu@yahoo.com.cn
作者简介:王伟,男,博士,讲师,研究方向:肿瘤基因治疗。
基金资助:
江苏省自然科学基金资助项目(BK2011624);江苏省高校长自然科学研究项目(10kjb310007);中央高校基本科研业务费专项资金项目(JKQ2009017);省级大学生实践创新训练计划项目

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

WANG Wei¹, WANG Yu², WANG Ruo-ning¹, XU Su-ying¹, DING Yang¹, ZHOU Jian-ping¹

¹Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, Jiangsu, China;
²Department of Pharmacology, Nanjing Medical University, Nanjing 210029, Jiangsu, China

Received:2011-10-10 Revised:2011-11-07 Online:2011-11-26 Published:2011-11-29

摘要/Abstract

摘要： 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p53 mRNA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,p53 mRNA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。

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

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 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.

Key words: Urocanic acid-modified chitosan, Wild-type p53, Gene transfection, Human hepatoma cell line, Cell cycle arrest

中图分类号:

R965.2

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.

WANG Wei, WANG Yu, WANG Ruo-ning, XU Su-ying, DING Yang, ZHOU Jian-ping. Study on effects of novel cationic polymer nonviral vector-mediated wild-type p53 gene transfection in vitro[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2011, 16(11): 1227-1233.

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