医学分子生物学杂志2011，Vol.8Issue(4)：313-317,5.DOI:10.3870/j.issn.1672-8009.2011.04.007
大肠埃希菌嘌呤核苷磷酸化酶基因载体的构建及对胰腺癌细胞的作用
Construction of Vector Expressing Escherichia Coli Purine Nucleoside Phosphorylase Gene and Study of Its Effect on Pancrease Cancer Cells
摘要
Abstract
Objective To construct recombinant retroviral vector pMSCV/PNP containing purine nucleoside phosphorylase ( PNP ) , determine the biological activity of PNP in pancrease cancer cells, with the aim to explore PNP in gene therapy. Methods PNP was amplified from Escherichia coli K12 by PCR and cloned into retroviral vector pMSCV. The recombinant vector pMSCV/PNP was analyzed with restriction endonucleases, PCR and sequencing. The retroviruses containing PNP were produced in 293 package cells , with viral titer was tested by FACS analysis. Pancreas cancer BXPC3 cells were infected with PNP containing retroviruses. The infected cells ( GFP positive ) were observed using fluorescence microscope and sorted by FACS. mRNA expression of PNP in BXPC-3/PNP cells was detected by RT-PCR. PNP activity was identified by MTT assay. Results PNP gene with 738bp was cloned into retroviral vector pMSCV, as verified by PCR and sequencing. Packaging cell line 293 produced high titer of pMSCV/PNP retroviruses ( 3. 6 107 U/ml ) . pMSCV/PNP infected BXPC-3 pancreas cancer cells expressed high level of PNP mRNA and responded to the cytotoxie and antiproliferative effects of MePdR ( 6-Methylpurine-2- Deoxyriboside ) in a concentration dependent manner, displaying 10％ survival at 1. 00 mg/L MePdR and up t0 100% cell death with the increase of MePdR. Conclusion PNP overexpression renders pancreas cancer cells BXPC-3/PNP sensitive to MePdR treatment.关键词
大肠埃希菌嘌呤核苷磷酸化酶/自杀基因/载体/胰腺癌Key words
purine nucleoside phosphorylase/ suicide gene/ vector/ pancrease cancer分类
生物科学引用本文复制引用
李文林,李克强,饶泽昌,石小玉,赵林..大肠埃希菌嘌呤核苷磷酸化酶基因载体的构建及对胰腺癌细胞的作用[J].医学分子生物学杂志,2011,8(4):313-317,5.基金项目
江西省自然科学基金(No.2007GZY1000),江西省卫生厅科技课题(No.20072017),浙江省医药卫生科学研究基金(No.2006) （No.2007GZY1000）
