Wang Hui, Zhang Jinxiang, Wu Heshui, Jiang Chunfang, Zheng Qichang, Li Zhuoya
Department of Medical Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2006;26(5):500-3. doi: 10.1007/s11596-006-0503-x.
In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-alpha) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37+/-8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-alpha released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.
为构建携带针对Toll样受体4（TLR4）mRNA的小发夹RNA（shRNA）和增强型绿色荧光蛋白（EGFP）报告基因的表达载体，并通过RNA干扰机制转染和表达靶向TLR4基因的shRNA，研究脂多糖（LPS）刺激诱导的RAW264.7细胞中细胞因子释放的抑制作用，使用了报告基因质粒pEGFP-C1（4.7 kb）和psiRNA-hHlneo（2979 bp）。从质粒psiRNA-hH1neo中克隆H1启动子和双Bbs I限制性内切酶位点，并在Mlu I限制性内切酶位点将它们重建到质粒pEGFP-C1中，形成含有Bbs位点和报告基因EGFP的质粒pEGFP-H1/siRNA。然后通过网络工具设计靶向TLR4基因的寡核发夹序列，并插入到质粒pEGFP-H1/siRNA中，形成质粒pEGFP-H1/TLR4-siRNA。将pEGFP-H1/TLR4-siRNA转染到RAW264.7细胞后，检测LPS刺激后细胞释放的肿瘤坏死因子-α（TNF-α）。结果表明，通过限制性内切酶分析证明构建的携带针对TLR4基因的发夹RNA和报告基因EGFP的pEGFP-H1/TLR4-siRNA是正确的。EGFP基因的表达为（50.37±8.23）%，转染质粒pEGFP-H1/TLR4-siRNA后，RAW264.7细胞释放的TNF-α水平下调。结论是，靶向TLR4基因的shRNA可以抑制LPS诱导的RAW264.7细胞释放TNF-α。
