慢病毒介导RNAi沉默大鼠脊髓神经元NF-κB p65基因的观察  

Lentivirus mediated RNA interference inhibits NF-κB p65 gene expression in spinal rat cord neurons

作　　者：罗剑刚[1] 孙涛[1] 林小雯[1] 李芸[1] 赵菲[1] 苗贵申 傅志俭[1] 

机构地区：[1]山东大学附属省立医院疼痛科,山东济南250021

出　　处：《山东大学学报（医学版）》2014年第3期23-26,32,共5页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81271346);山东省自然科学基金(ZR2010HM097)

摘　　要：目的 构建NFκBp65基因RNA干扰（RNAi）慢病毒载体并转染体外培养的大鼠脊髓神经元,观察其沉默效率。方法 针对大鼠NFκBp65基因特异性序列,设计3条NFκBp65siRNA序列及1条阴性对照序列,合成包含各正反义靶序列的互补DNA链,退火形成双链DNA,插入到经BamHI和EcoRI酶切后的pFUGW慢病毒载体中,PCR筛选阳性克隆,DNA测序鉴定。与慢病毒包装质粒pHelper1.0、pHelper2.0通过lipofectamine2000共转染至包装细胞293T,经滴度测定后转染体外培养的大鼠脊髓神经元。实验分为5组：阴性对照组、LVNC组、LVshNFκBp651组、LVshNFκBp652组和LVshNFκBp653组,观察转染效率,Westernblotting法检测慢病毒表达载体对神经元NFκBp65基因的沉默效应。结果 测序结果证实合成的寡核苷酸链插入正确,重组载体构建成功,测定病毒滴度为108～9TU/mL,慢病毒重组体能够成功转染大鼠脊髓神经元,转染效率大于90%,与阴性对照组或LVNC组比较,LVshNFκBp651组和LVshNFκBp652组NFκBp65蛋白的表达明显下调（P均〈0.01）,而LVshNFκBp653组无明显下调（P〉0.05）。结论 成功构建了大鼠神经元NFκBp65基因的RNAi慢病毒载体,它可使大鼠脊髓神经元NFκBp65基因表达下调。Objective To construct a lentiviral vector for RNA interference of rat NF-κB p65 gene and transfect it into spinal neurons to observe its silencing efficiency. Methods Three siRNA sequences and a negative control sequence were designed according to NF-κB p65 gene sequence of rats. The complementary DNA containing both sense and anti- sense DNA oligos of the targeting sequence was synthesized and cloned into the pFU-GW vector, which were digested by BamH I and EcoR I. The recombined vector was confirmed by PCR and DNA sequencing and cotransfected with pHelperl. 0 and pHelper 2.0 packaging plasmids into 293T cells by use of Lipofectamine 2000, then the virus titer was measured. Finally, the recombinant lentivirus was transfected into spinal neurons, which were divided into five groups： negative control group, LV-NC group, LV-shNF-κB p65-1group, LV-shNF-κB p65-2 group and LV-shNF-κB p65-3 group. The transfection efficiencies were observed in each group. Results DNA sequencing results demonstrated that the inserted sequences were correct. The titer of virus was 10^8-9 TU/mL. Recombinant lentivirus could be successfully transfected into spinal neurons with the transduction rate higher than 90%. Compared with negative control or LV-NC groups, LV-shNF-κB p65-1 or LV-shNF-κB p65-2 groups showed a lower expression level of NF-κBp65 protein（ all P 〈 0. 01 ）, while LV-shNF-κB p65-3 group didn＇ t （ P 〉 0.05 ）. Conclusion The lentivirus RNAi vector targeting rat spinal neuronal NF-κB p65 gene has been constructed successfully. It may down-regulate NF-κB p65 expression in spinal neurons.

关 键 词：RNA干扰 核转录因子κBp65 慢病毒 神经元 大鼠 

分 类 号：R745[医药卫生—神经病学与精神病学]