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

摘要

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 a 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.%肿瘤坏死因子α (tumor necrosis factor α,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-α mRNA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.