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

Abstract

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

CLC Number:

R965.2

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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