郑州大学学报（医学版）2012，Vol.47Issue(5)：629-632,4.DOI:10.3969/j.issn.1671-6825.2012.05.012
TNF-α基因siRNA真核表达载体的构建及其对A549细胞TNF-α和TGF-β1表达的影响
Construction of eukaryotic expression vectors of siRNA against TNF-α gene and their silencing effects on TNF-α and TGF-β1 in A549 cells
摘要
Abstract
Aim: To clone the eukaryotic expression vectors of small interfering RNA ( siRNA ) against TNF-a gene and to evaluate their silencing effects on TNF-a and TGF-β1 in A549 cells. Methods: According to TNF-a gene ( NM_ 000594 ) sequence of GenBank, using RNAi Designer software to design the siRNA specific target sequence ( siTNF-α ) and the unrelated control sequence ( siCon ) for TNF-α. The annealing products were recombined into pRNAT-U6. 1, PCR and DNA sequencing confirmed the insertion sequence. The recombinant plasmids ( pRNAT-U6. l-siTNF-a,pRNAT-U6. 1-siCon ) were transfected to the A549 cells with lipofectamine,and the expressions of TNF-α, TGF-β1 mRNA and protein in cells detected by Real time PCR and Western blot methods. Results: PCR and DNA sequencing confirmed the vectors were consistent;the expressions of TNF-α, TGF-β1 mRNA and protein in the cells transfected pRNAT-U6. 1-siTNF-a were markedly lower than the other three controls( FmRNA = 478. 663 ,4. 081 ;Fprotein = 123. 420,6. 312, all P < 0. 05 ). Conclusion:The TNF-a siRNA eukaryotic expression vector is constructed successfully, which could silence the expression of TNF-α in A549,as well as the expression of TGF-β1.关键词
肿瘤坏死因子α/转化生长因子-β1/RNA干扰/肺腺癌/A549细胞Key words
TNF-α/ TGF-β1 /RNA interference/lung adenocarcinoma/A549 cell分类
医药卫生引用本文复制引用
张立英,刘绍霞,赵国强,张国俊..TNF-α基因siRNA真核表达载体的构建及其对A549细胞TNF-α和TGF-β1表达的影响[J].郑州大学学报（医学版）,2012,47(5):629-632,4.基金项目
河南省杰出青年基金资助项目 094100510014 （）
