Dicer-substrate siRNA inhibits tumor necrosis factor alpha secretion in Kupffer cells in vitro: in vivo targeting of Kupffer cells by siRNA-liposomes.
Dicer-substrate siRNA inhibits tumor necrosis factor alpha secretion in Kupffer cells in vitro: in vivo targeting of Kupffer cells by siRNA-liposomes.
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Dicer 底物 siRNA 体外抑制 Kupffer 细胞肿瘤坏死因子 α 分泌：体内 siRNA 脂质体靶向 Kupffer 细胞。
DOI：
10.1016/j.phrs.2011.09.001
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发表时间：
2012
影响因子：
9.3
通讯作者：
Ponnappa,BiddandaC
中科院分区：
文献类型：
--
作者：
Pichu,Sivakamasundari;Krishnamoorthy,Swapna;Zhang,Bi;Jing,Yawu;Shishkov,Andrei;Ponnappa,BiddandaC
肿瘤坏死因子-α在许多炎症性疾病的发病机制中起重要作用。用抗体或反义寡核苷酸中和肿瘤坏死因子-α，减轻疾病症状。在本研究中，我们引入了新一代的基因沉默分子，即小干扰RNA(SiRNAs)来降低肿瘤坏死因子-α。虽然19-21bp的siRNAs被普遍使用，但据报道，较长的siRNAs具有更高的效率。在此，我们报告了针对肿瘤坏死因子-α基因的27聚体底物小干扰RNA的鉴定。将5个27聚体siRNA载体(si27-1、si27-2、si27-3、si27-4和si27-5)导入原代培养的大鼠枯否细胞，24小时后用脂多糖(0.1μg/ml)刺激细胞分泌肿瘤坏死因子-RNA。用双抗体夹心法测定培养上清中释放的肿瘤坏死因子α。在5个si27构建体中，si27-3对肿瘤坏死因子-α的抑制作用最强。在10 nm处，si27-3对肿瘤坏死因子-α的抑制率为80%，而21-聚体(Ssl3)的抑制率为60%。在阴离子脂质体包裹后，连续两天静脉注射100μg/kg体重的Si27-3可抑制50%的肿瘤坏死因子-α的分泌。这些数据证明了一种高效的小干扰RNA制剂的鉴定，该制剂可用于治疗肿瘤坏死因子-α介导的疾病。
Tumor necrosis factor alpha (TNF-α) plays a major role in the pathogenesis of many inflammatory diseases. Neutralizing TNF-α by antibodies or antisense oligodeoxynucleotides, alleviate disease symptoms. In this study, we introduce the new generation of gene-silencing molecules, namely the small interfering RNAs (siRNAs) to reduce TNF-α. Although siRNAs of 19–21bp are commonly used, it is reported that longer siRNAs have much higher efficacies. Here, we report the identification of a 27-mer Dicer-substrate siRNA (DsiRNA) against TNF-α mRNA. Primary cells of rat Kupffer cells were transfected with five 27-mer siRNA constructs (si27-1, si27-2 si27-3, si27-4 and si27-5) for 24h, following which, TNF-α secretion was induced by exposure to LPS (0.1μg/ml) for 2h. TNF-α released to the medium was measured by ELISA. Of the five si27 constructs, si27-3 had the highest inhibitory effect on TNF-α secretion. At 10nM, si27-3 inhibited TNF-α secretion by 80% compared to a 60% inhibition by a 21-mer (SSL3). Following encapsulation in anionic liposomes, si27-3 at 100μg/kg body weight, on two successive days by intravenous administration, inhibited the secretion of TNF-α by 50%. These data demonstrate the identification of a highly efficacious siRNA formulation, which can be used in the treatment of TNF-α mediated diseases.