SiRNA抑制大鼠背根神经节神经元电压依赖型钠通道Nav1.8表达的实验研究  

Small interfering RNA inhibits voltage-gated sodium channel Nav1.8 expression in cultured dorsal root ganglion neurons

作　　者：刘艳红[1] 张宏[1] 徐龙河[1] 

机构地区：[1]解放军总医院麻醉手术中心,北京100853

出　　处：《北京医学》2013年第8期653-656,F0002,共5页Beijing Medical Journal

基　　金：国家自然科学基金(30901398;30300327)

摘　　要：目的应用小片段干扰RNA(SiRNA)转染原代培养背根神经节(DRG)神经元,观察SiRNA干扰DRG神经元基因Nav1.8表达的有效性。方法根据RNA干扰的原理及SiRNA的设计原则,设计2个长度为21bp的以Nav1.8基因为靶点的SiRNA(SiRNAa和SiRNAb),另选择相同长度的无义双链RNA作为阴性对照RNA。T7RNA聚合酶的作用下体外转录合成SiRNA,阳离子脂质体转染试剂将SiRNA导入体外培养DRG神经元,于转染后48h提取RNA,采用RT-PCR和荧光定量PCR检测Nav1.8基因的表达情况。结果在靶向Nav1.8的两个SiRNA序列中,SiRNAa转染神经元后导致Nav1.8基因表达显著降低(48.32±6.84)%,SiRNAb使Nav1.8表达降低(23.32±3.19)%,P均<0.01,阴性对照SiRNA对Nav1.8的表达不产生影响。结论利用体外转录合成的SiRNA成功转染原代培养的DRG神经元,并诱导产生内源性Nav1.8基因的特异性表达抑制。具有较高干扰效应的SiRNA片段可应用于抑制Nav1.8基因表达,进而研究Nav1.8的功能及在疼痛治疗中的作用。Objective To evaluate the efficiency of the RNA interference effect on voltage-gated sodium channel Navl.8 by transfecting small interference RNA （siRNA） into primary cultured dorsal root ganglion neurons. Methods Based on the general roles of RNAi and siRNA design, 2 pairs of siRNAs were synthesized targeting Navl.8 （named SiRNAa and SiRNAb） by in vitro transcription with T7 RNA polymerase. A mismatched double-strand RNA was choosen as negative control RNA. SiRNA were transfected into primary cultured dorsal root ganglion neurons with cationic lipid based transfection reagent. Expression of Navl.8 was determined with RT-PCR and Q-PCR 48 h after the transfecion. Results In the 2 pairs of SiRNA targeting Navl.8, SiRNAa induced significant decrease in Navl.8 expression [（48.32±6.84）%] compared with control, while SiRNAb pro-duced a （23.32±3.19）% decrease in Navl.8 expression compared with control. Negative control RNA failed to cause any change in the expression of Navl.8. Conclusion SiRNA synthesized by in vitro transcription can inhibit endogenous Navl.8 gene ex-pression in cultured primary DRG neurons. SiRNA with higher interfering effect can be used to inhibit Navl.8 expression. It can be used to study the function of Navl.8 and its role in pain treatment.

关 键 词：RNA干扰 电压依赖型钠通道Nav1.8 背根神经节 神经元 

分 类 号：R614[医药卫生—麻醉学]