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

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.