Short hairpin RNA-mediated selective knockdown of NaV1.8 tetrodotoxin-resistant voltage gated sodium channel in dorsal root ganglion neurons
Short hairpin RNA-mediated selective knockdown of NaV1.8 tetrodotoxin-resistant voltage gated sodium channel in dorsal root ganglion neurons
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DOI：
10.1097/00000542-200510000-00022
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发表时间：
2005-10-01
期刊：
影响因子：
8.8
通讯作者：
Yang, J
中科院分区：
文献类型：
--
作者：
Mikami, M;Yang, J
背景：电压门控钠通道由一系列密切相关的蛋白质组成，每种蛋白质都具有不同的生理和病理功能。Nav1.8是电压门控钠通道的一种异构体，与炎症性和神经性疼痛的发病机制有关，但目前，对于任何电压门控钠通道都没有异构体特异性抑制剂。作者探讨了短发夹RNA介导的Nav1.8表达选择性敲低的可能性。
方法：设计用于转录靶向Nav1.8的短发夹RNA的DNA构建体，并将其整合到重组慢病毒中。利用蛋白质印迹法、免疫组织化学、逆转录聚合酶链反应和膜片钳电生理学技术，在蛋白质、信使RNA和功能水平上检测病毒诱导的Nav1.8选择性敲低情况。
结果：用表达短发夹RNA的慢病毒转导稳定表达Nav1.8的HEK293细胞或原代背根神经节神经元，导致Nav1.8蛋白和信使RNA浓度降低。全细胞膜片钳记录证实Nav1.8介导的电流密度降低，而其他生物物理特性无变化。
结论：通过慢病毒递送短发夹RNA可实现背根神经节神经元中Nav1.8表达的选择性敲低。这种方法可能为控制神经元过度兴奋和病理性疼痛提供一种新的基因治疗方法。
Background: Voltage-gated sodium channels comprise a family of closely related proteins, each subserving different physiologic and pathologic functions. NaV1.8 is an isoform of voltage-gated sodium channel implicated in the pathogenesis of inflammatory and neuropathic pain, but currently, there is no isoform-specific inhibitor of any voltage-gated sodium channels. The authors explored the possibility of short hairpin RNA-mediated selective knockdown of NaV1.8 expression.Methods: DNA constructs designed to transcribe short hairpin RNA targeting NaV1.8 were created and incorporated into recombinant lentiviruses. The virus-induced selective knockdown of NaV1.8 was examined at the protein, messenger RNA, and functional levels using Western blot, immunohistochemistry, reverse-transcription polymerase chain reaction, and patch clamp electrophysiology.Results: Transduction of HEK293 cells stably expressing NaV1.8 or primary dorsal root ganglion neurons with lentivirus expressing short hairpin RNA resulted in the knockdown of NaV1.8 protein and messenger RNA concentrations. Whole cell patch clamp recordings confirmed decrease in the NaV1.8-mediated current density without changes in other biophysical properties.Conclusions: A selective knockdown of NaV1.8 expression in dorsal root ganglion neurons can be attained by short hairpin RNA delivered with lentivirus. This method may provide a new gene therapy approach to controlling neuronal hyperexcitability and pathologic pain.