嗜酸粒细胞亲合素N端突变体重组腺病毒在小鼠变应性鼻炎中的作用  

Effects of recombinant eotaxin N terminal mutant (Met/Eotaxin) adenovirus on allergic rhinitis in mice

作　　者：朱瑾[1] 韩德民[1] 周锦川[2] 贺伟峰[2] 张小容[2] 郑红[2] 吴军[2] 

机构地区：[1]首都医科大学附属北京同仁医院耳鼻咽喉头颈外科 [2]重庆第三军医大学烧伤研究所

出　　处：《中华耳鼻咽喉头颈外科杂志》2006年第3期176-180,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家自然科学基金资助(30200313)

摘　　要：目的获取人嗜酸粒细胞亲合素(Eotaxin)N端突变体(Met/Eotaxin)重组腺病毒,探讨其在小鼠变应性鼻炎中的作用。方法从人外周血单个核细胞总RNA中克隆人400bp的Eotaxin基因;通过N端点突变获取Met/Eotaxin基因,构建pAdtrack-CMV-Met/Eotaxin腺病毒穿梭质粒;将该质粒在大肠杆菌BJ5183中与腺病毒基因组质粒进行同源重组;将Met/Eotaxin重组腺病毒载体在AD293细胞中包装、扩增获取Met/Eotaxin重组腺病毒(Ad-Met/Eotaxin);采用聚合酶链式反应(polymerase chain reaction,PCR)和蛋白印迹杂交(Western blot)对Met/Eotaxin重组腺病毒的正确性进行鉴定。采用卵清蛋白(ovalbumin,OVA)致敏和激发诱导小鼠变应性鼻炎,40只小鼠随机分为变应性鼻炎组和Ad-Met/Eotaxin组。Ad-Met/Eotaxin组在每次激发前30min予以每只小鼠2ml滴度为5×109pfu/ml的Ad-Met/Eotaxin腹腔注射。比较二组动物变应性鼻炎症状和鼻黏膜形态学改变,并在荧光显微镜下观察腺病毒转染效率标志物———绿色荧光蛋白在鼻黏膜等组织中的表达。结果PCR和Western blot结果显示Ad-Met/Eotaxin构建正确;Ad-Met/Eotaxin腹腔注射后可高效转染小鼠鼻黏膜;Ad-Met/Eotaxin可显著减轻小鼠变应性鼻炎症状以及鼻黏膜形态学改变。结论Ad-Met/Eotaxin具有阻断小鼠变应性鼻炎的作用。Objective To obtain the recombinant Eotaxin N Terminal Mutant （Met/Eotaxin） advenvirus, and to study its role in allergic diseases. Methods A 400 bp DNA fragment of Eotaxin was cloned from human peripheral blood monocyte （PBMC） , and the Met/Eotaxin DNA fragment was obtained by NH2-terminal mutant. Then, this DNA fragment was inserted into AdEasy transfer vector -pAdtrack-CMV plasmid. The plasmid pAdtrack-CMV-Met/Eotaxin was then linearized with Pine I and co-transformed into the Ecoli strain BJ5183 together with pAdEasy-1, the viral DNA plasmid. The recombinant Met/Eotaxin adenovirus vector was then transfected into 293 cells to produce and amplify viral particles. The allergic rhinitis in mice was induced by sensitizating and challenging with ovalbumin （OVA）. Fourty mice were equally divided into allergic rhinitis group （AR group） and Ad-Met/Eotaxin group. 2 ml Ad-Met/Eotaxin with titer of 5 × 10^9 pfu/ml was intraperitoneal injected in each mice in Ad-Met/Eotaxin group 30 min before each challenge. The symptom and the morphological change of nasal mucosa were compared in those two groups. The enhanced green fluorescence protein （EGFP）expression corresponding to the efficiency of transfection in the Ad-Met/Eotaxin group was observed under fluorescence microscope. Resolts The results of sequencing and PCR showed that the Met/Eotaxin gene recombinant replication-deficient adenovirus was successfully constructed. The adenovirus marker of EGFP could be observed in a higher intensity after Ad- Met/Eotaxin intraperitoneal injection. Ad-Met/Eotaxin adenovirus significantly alleviated the symptoms and nasal histological changes of allergic rhinitis. Conclusion Ad-Met/Eotaxin could be transfected efficiently with high titer in nasal mucosa of mice, and could alleviate the symptoms of allergic rhinitis in mice.

关 键 词：趋化因子 突变体 鼻炎 变应性 

分 类 号：R765.21[医药卫生—耳鼻咽喉科]