Kako Keiko, Nishikawa Makiya, Yoshida Hiroyuki, Takakura Yoshinobu
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
J Pharm Sci. 2008 Aug;97(8):3074-83. doi: 10.1002/jps.21254.
To examine the effects of inflammatory response to plasmid DNA (pDNA) on transgene expression, serum tumor necrosis factor-alpha (TNF-alpha) was measured after intravenous injection of pDNA or calf thymus DNA (CT DNA) in the naked or complexed form with cationic liposomes (lipoplex). pDNA with many CpG motifs induced TNF-alpha production regardless of the forms. No significant TNF-alpha production was detected when CT DNA or methylated pDNA was injected. Clodronate liposomes and dexamethasone were used to deplete phagocytes or to inhibit inflammatory responses, respectively. Transient depletion of phagocytes, such as liver Kupffer cells and splenic macrophages, by clodronate liposomes slightly altered the tissue distribution of (32)P-pDNA lipoplex, but significantly reduced the TNF-alpha production and transgene expression. Dexamethasone significantly inhibited the initial transgene expression, but increased the duration of the expression slightly. Use of NF-kappaB activity-dependent plasmid vector suggested that the inhibition of NF-kappaB activation is involved in the reduced expression by these treatments. These findings indicate that tissue macrophages are closely involved in the CpG motif-dependent TNF-alpha production. It is also suggested that TNF-alpha activates NF-kappaB and increases transgene expression by pDNA having many NF-kappaB binding sites, but TNF-alpha also reduces transgene expression at later time periods, leading to short-term transgene expression.
为了研究炎症反应对质粒DNA（pDNA）转基因表达的影响，在静脉注射裸pDNA或与阳离子脂质体复合（脂质体复合物）的pDNA或小牛胸腺DNA（CT DNA）后，测量血清肿瘤坏死因子-α（TNF-α）水平。含有许多CpG基序的pDNA无论何种形式均能诱导TNF-α产生。注射CT DNA或甲基化pDNA时未检测到明显的TNF-α产生。氯膦酸脂质体和地塞米松分别用于清除吞噬细胞或抑制炎症反应。氯膦酸脂质体对吞噬细胞（如肝库普弗细胞和脾巨噬细胞）的短暂清除略微改变了（32）P-pDNA脂质体复合物的组织分布，但显著降低了TNF-α产生和转基因表达。地塞米松显著抑制初始转基因表达，但略微延长了表达持续时间。使用依赖NF-κB活性的质粒载体表明，这些处理导致的表达降低与NF-κB激活的抑制有关。这些发现表明组织巨噬细胞密切参与了CpG基序依赖性TNF-α的产生。还表明TNF-α激活NF-κB并通过具有许多NF-κB结合位点的pDNA增加转基因表达，但TNF-α在后期也会降低转基因表达，导致转基因的短期表达。
