Gene knockdown with lentiviral vector-mediated intrathecal RNA interference of protein kinase C gamma reverses chronic morphine tolerance in rats
Gene knockdown with lentiviral vector-mediated intrathecal RNA interference of protein kinase C gamma reverses chronic morphine tolerance in rats
复制标题
慢病毒载体介导的蛋白激酶 C γ 鞘内 RNA 干扰基因敲除可逆转大鼠慢性吗啡耐受
DOI：
10.1002/jgm.1514
复制
发表时间：
2010-11-01
影响因子：
3.5
通讯作者：
Guo, Qulian
中科院分区：
文献类型：
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作者：
Song, Zongbin;Zou, Wangyuan;Guo, Qulian
背景虽然吗啡是一种广泛使用的阿片类镇痛药，但吗啡耐受（MT）限制了药物的使用，因为它需要大剂量。蛋白激酶C（PKC），特别是PKC γ亚型，被认为在MT的发生发展中起关键作用。由于RNA干扰为研究基因功能提供了一种强有力的方法，并且慢病毒递送系统已被批准用于人类，本研究检测了对吗啡耐受的大鼠，以确定鞘内注射PKC γ短发夹RNA的慢病毒载体是否（LV-shPKC γ）下调PKC γ基因的表达，逆转MT。方法鞘内注射吗啡（10 μ g b.i.d.）连续六天。合成慢病毒介导的短发夹RNA（shRNA）系统，将PKC γ shRNA递送到MT大鼠的脊髓。以机械和热缩足阈测定吗啡的镇痛作用。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法（Western blotting）分别检测PKC γ mRNA和蛋白的表达。单次注射LV-shPKC γ可显著逆转吗啡镇痛耐受。与对照组相比，PKC γ mRNA和蛋白水平显着下调LV-shPKC γ group.Conclusions单次注射LV-shPKC γ逆转MT通过减少PKC γ在脊髓中的表达。这些发现表明，使用LV-shPKC γ可能是MT治疗的潜在策略。版权所有（C）2010约翰威利父子有限公司
Background Although morphine is a widely used opioid analgesic, morphine tolerance (MT) has limited the use of the drug because it creates the necessity for high doses. Protein kinase C (PKC), especially the PKC gamma isoform, is considered to play a key role in the development of MT. Because RNA interference provides a powerful method for the investigation of gene function, and lentiviral delivery systems have been approved for human use, this present study examined rats tolerant to morphine to determine whether an intrathecal injection of a lentiviral vector of PKC gamma short hairpin RNA (LV-shPKC gamma) down-regulated the expression of the PKC gamma gene and reversed MT.Methods MT was induced by intrathecal morphine (10 mu g b.i.d.) for six consecutive days. A lentiviral-mediated short hairpin RNA (shRNA) system was synthesized to deliver the PKC gamma shRNAs to the spinal cord of the rats with MT. Mechanical and thermal paw withdrawal threshold were assessed to determine the analgesic effects of morphine. Expression of PKC gamma mRNA and protein was determined by reverse transcriptase-polymerase chain reaction and western blotting analysis, respectively.Results The chronic administration of morphine induced a stabilized analgesic tolerance. A single injection of LV-shPKC gamma significantly reversed morphine antinociceptive tolerance. Compared to the control group, PKC gamma mRNA and protein levels were dramatically down-regulated in the LV-shPKC gamma group.Conclusions A single injection of LV-shPKC gamma reversed MT by reducing the expression of PKC gamma in the spinal cord. These findings indicate that the use of LV-shPKC gamma might be a potential strategy for therapy in MT. Copyright (C) 2010 John Wiley & Sons, Ltd.