肿瘤坏死因子α突变型斑马鱼在海分枝杆菌感染中的应用研究  

Application of tumor necrosis factorα mutant zebrafish in Mycobacterium marinuminfection

作　　者：谢玲玲[1] 王风华[2] 王德成[3] 赵超[1] 彭刚[2] 牛辰[1] 

机构地区：[1]复旦大学基础医学院医学分子病毒学教育部/卫生部重点实验室,上海200032 [2]复旦大学脑科学研究院,上海200032 [3]三峡大学医学院,宜昌443002

出　　处：《微生物与感染》2017年第3期156-163,共8页Journal of Microbes and Infections

基　　金：上海市科学技术委员会科研计划项目(14140902900)

摘　　要：肿瘤坏死因子α(tumor necrosis factorα,TNF-α)作为重要的炎性细胞因子,在类风湿关节炎、感染性休克等疾病的发病中起重要作用。为探究TNF-α在宿主抵抗海分枝杆菌感染中的作用,本研究采用成簇的规律间隔的短回文重复序列及其相关蛋白9[clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9]技术,使野生型斑马鱼TNF-α基因发生突变,成功构建并筛选出含有移码突变的TNF-α突变型(TNF-α-/-)斑马鱼。利用原位杂交技术,发现TNF-α-/-斑马鱼中TNF-αmRNA表达水平显著降低。与野生型斑马鱼相比,海分枝杆菌在TNF-α-/-斑马鱼体内扩散和增殖更显著。结果提示,本研究成功构建TNF-α-/-斑马鱼,有助于深入探究TNF-α在斑马鱼抵抗海分枝杆菌感染中的作用及其免疫调节机制。Tumor necrosis factor α（TNF-α） is an important inflammatory cytokine implicated in the pathogenesis of rheumatoid arthritis, septic shock and other diseases. In order to investigate the functional role of host TNF-α in Mycobacterium marinum （M. marinum） infection, clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated protein 9 （Cas9） （CRISPR/Cas9） technology was utilized to mutate the TNF-α gene in wild type （WT） zebrafish. A zebrafish containing α frame-shift mutation was successfully constructed, isolated, and named as TNF-α -/-. It was found that the expression of TNF-α mRNA was significantly reduced in TNF-α-/- zebrafish by in situ hybridization （ISH）. The proliferation of M. marinum in TNF-α-/- zebrafish was significantly elevated compared to WT zebrafish. The construction of TNF-α-/- zebrafish will allow further in-depth study on the function of TNF-α in mycobacterial infection and related immune regulation.

关 键 词：成簇的规律间隔的短回文重复序列及其相关蛋白9 肿瘤坏死因子Α 突变型斑马鱼 原位杂交 细菌感染 

分 类 号：R378.91[医药卫生—病原生物学]