Hartmann G, Krug A, Eigler A, Moeller J, Murphy J, Albrecht R, Endres S
Medizinische Klinik, Klinikum Innenstadt of the Ludwig-Maximilians-University, Munich, Germany.
Antisense Nucleic Acid Drug Dev. 1996 Winter;6(4):291-9. doi: 10.1089/oli.1.1996.6.291.
Recent clinical studies using neutralizing antibodies point to a key role for tumor necrosis factor-alpha (TNF-alpha) in chronic inflammatory diseases. Antisense technique is a recent approach aiming at inhibition of single proteins. Previously, we described nonspecific induction of TNF by phosphorothioate oligonucleotides. In this study, we established an in vitro model that allows specific inhibition of TNF synthesis, bypassing TNF induction. Freshly isolated human monocytes were incubated with oligonucleotides and the cationic lipid lipofectin in different ratios. TNF synthesis was stimulated with lipopolysaccharide and quantified by a specific radioimmunoassay (RIA). Among all sequences tested, one of the antisense oligonucleotides complementary to the translation initiation region of TNF mRNA (5'-CAT GCT TTC AGT CAT-3') revealed highest efficacy. At 2 microM, the antisense oligonucleotide inhibited TNF synthesis by up to 79%. A concentration as low as 250 nM of the antisense oligonucleotide was effective. Scrambled controls and controls with different, defined degrees of mismatches confirmed a sequence-specific action. Examination with confocal fluorescence microscopy showed a marked difference comparing lipofectin-mediated vs. spontaneous uptake. This study defines criteria that from the prerequisite necessary for design and application of antisense oligonucleotides against TNF in vivo.
近期使用中和抗体的临床研究表明，肿瘤坏死因子-α（TNF-α）在慢性炎症性疾病中起关键作用。反义技术是一种旨在抑制单一蛋白质的最新方法。此前，我们描述了硫代磷酸酯寡核苷酸对TNF的非特异性诱导作用。在本研究中，我们建立了一种体外模型，该模型能够绕过TNF诱导，特异性抑制TNF的合成。将新鲜分离的人单核细胞与寡核苷酸和阳离子脂质lipofectin按不同比例孵育。用脂多糖刺激TNF合成，并通过特异性放射免疫测定（RIA）进行定量。在所有测试序列中，与TNF mRNA翻译起始区域互补的反义寡核苷酸之一（5'-CAT GCT TTC AGT CAT-3'）显示出最高的效力。在2 microM时，反义寡核苷酸对TNF合成的抑制率高达79%。低至250 nM的反义寡核苷酸浓度就有效。随机对照和具有不同程度错配的对照证实了序列特异性作用。共聚焦荧光显微镜检查显示，lipofectin介导的摄取与自发摄取相比有显著差异。本研究确定了体内设计和应用针对TNF的反义寡核苷酸所需的前提条件标准。
