核因子-κB特异性的RNA干扰腺病毒的构建及其对枯否细胞内毒素反应的影响  

Construction of the adenovirus expressing NF-κB SiRNA in rats and its effect on Kupffer cells reaction to LPS

作　　者：吴飞翔[1] 吕欣[1] 孙玉明[1] 俞卫锋[1] 黄盛东 杨立群[1] 徐学武[1] 袁扬 

机构地区：[1]第二军医大学东方肝胆外科医院麻醉科,上海200438 [2]解放军胸心外科研究所

出　　处：《中华实验外科杂志》2009年第2期177-180,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30700788）

摘　　要：目的构建针对大鼠核因子（NF）-κB的腺病毒，采用RNA干扰方法观察其对内毒素诱导的大鼠肝枯否细胞（κC）NF—κB抑制和肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6释放的影响。方法合成双链寡核苷酸siNF—κB克隆入pShuttleHl载体，取0．5μg线性化后的pShuttleHlsiNF—κB电转化BJ5183感受态细菌获取重组腺病毒骨架质粒；取线性化的重组质粒4μg利用Lipo—fectamine^TM2000转染骨架质粒至HEκ293细胞获取重组腺病毒Ad—siNF—κB；TCIDS0法测定病毒滴度；腺病毒以MOI=10感染原代培养大鼠肝枯否细胞，观察对内毒素诱导的NF—κB抑制和TNF-κB、IL-α释放的影响。结果目的基因成功克隆入腺病毒，病毒滴度为5．32×10^9pfu／ml。转染腺病毒Ad—siNF—κB后的内毒素诱导的肝KC及NF—κBmRNA及TNF-α、IL-6的表达均明显减弱。结论成功构建表达NF—κBsiRNA的腺病毒，具有良好的抑制NF—κB和TNF-α、IL-6的作用。Objective To construct the incompetent-replication adenovirus expressing NF-κB siRNA in rats and identify its effect on κupffer ceils reaction to lipopolysaccharide （LPS） in vitro. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was cloned into the pShuttleH1 vector. Linearized pShuttleHl-siNF-κB of 0.5 μg was transformed into E. coli BJ5183 cells containing backbone plasmid pAdEasy-1 by electroporation. The recombinant plasmid of 4 μg was transfected into 293 cells to package the adenovirus Ad-siNF-κB. The titers of adenovirus were determined using the specific 50% tissue culture infection dosage （TCID50） method. After virus infected the cultured κupffer cells with MOI = 10, the effect on LPS-induced NF-κB mRNA and TNF-α, IL-6 expression was observed. Results It was identified that the sequence of gene was correctly inserted into the genome of virus. The titer of recombinant adenovirus was 5.32 ×10^9 pfu/ml. NF-κB mRNA and TNF-α, IL-6 expression was greatly reduced after virus infection. Conclusion The recombinant adenovirus expressing NF-κB siRNA in rats were successfully constructed, which probably can be further used in research on anti-inflammation effect in vivo.

关 键 词：NF—κB RNA干扰 腺病毒 肿瘤坏死因子-α 枯否细胞 

分 类 号：R[医药卫生]