摘要
目的 通过构建IκBα基因的腺病毒表达载体 ,并用急性相反应蛋白SAA3 启动子来指导目的基因IκBα的表达 ,体外观察在炎性刺激后IκBα蛋白的诱导性表达特性及对NF κB活性的调控作用 ,对炎症介质产生的抑制效应。方法 采用DNA重组与同源重组技术 ,构建含只响应病理信号的启动子 急性相反应蛋白启动子SAA3 、目的基因IκBα的炎症诱导性复制缺陷型重组腺病毒载体 (AdIκBα)。应用AdIκBα,选择在炎症反应中具有代表意义的巨噬细胞 ,观察AdIκBα在体外培养巨噬细胞U93 7中诱导性表达IκBα(Westernblot)、对NF κB活性的调控 (EMSA)及体外效应。结果 ①成功构建炎症诱导性IκBα表达质粒 (pAdpLplSAA3 NLSIκBα) ,该表达框含SAA3 启动子 ,SV40 大T抗原核定位信号 (NLS) ,IκBαcDNA和ployA ;②pAdpLpLSAA3 NLSIκBα质粒与pJM1 7同源重组 ,脂质体 (DOTAP)介导 ,共转染入 2 93细胞。PCR法筛选阳性克隆 ,获得重组腺病毒 (AdIκBα) ;③用 2 93细胞扩增AdIκBα;采用反复冻融法纯化腺病毒 ,获得高滴度 (5× 1 0 1 0 pfu/ml)重组腺病毒 ;④体外实验 ,用 50MOIAdIκBα预感染体外培养的U93 7细胞 ,再用LPS攻击后 ,该腺病毒诱导性表达IκBα蛋白 (Westernblot) ,且表达的IκBα能抑制NF κB的活化 (EMSA)
Objective To investigate the expression of IκBα triggered by an acute phase protein promoter SAA 3 (Serum Amyloid A3 gene ) after adenoviral transfer in cultured U 937 cells and its inhibitory effects on the activation of NF κB in response to inflammatory stimuli, and on the production of other cytokines. Methods A recombinant adenovirus (Ad IκBα) encoding IκBα was constructed by means of homorecombination and then 50 MOI Ad IκBα were used to infect the cultured U 937 cells, which were challenged with LPS (10 μg/ml) 24 h later. After that, the whole cell proteins and nuclear proteins were extracted respectively and detected with western blot. The NF κB/DNA binding activity of nuclear proteins was determined with electrophoretic mobility shifty assay (EMSA). The TNF α and IL 6 productions in the culture supernatant were measured with ELISA. Results ①The inflammatory induced expressing plasmid (pAdpLpL SAA 3 NLS IκBα) encoding SAA 3 promoter, SV40 large T nucleus location signal (NLS), IκBα cDNA and polyA was constructed successfully.②DOTAP mediated gene transfer was used to co transfect the pAdpLpL SAA 3 NLS IκBα shuttle vector and the recombination plasmid pJM17 into 293 cells. The positive clones were obtained by using PCR to obtain the adenovirus of IκBα gene (Ad IκBα).③The recombinant virus was amplified in 293 cells and purified with freeze thaw method for a higher titer (5×10 10 virus /ml).④IκBα protein was induced rapidly in the cultured U 937 cells after LPS challenge and inhibited the NF κB ctivation and reduced the productions of TNF α and IL 6. Conclusion Adenoviral transfer of IκBα can modulate the production of inflammatory cytokines by inhibiting the activation of NF κB in U 937 cells after LPS challenge, indicating its therapeutic effects in inflammatory reactions
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2001年第12期1386-1389,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目 ( 3 9770 717)
