Tet1对宿主抗海分枝杆菌感染的意义与机制研究

Significance and mechanism of Tet1 against Mycobacterium marinum infection in mice

目的探讨宿主Tet1(ten-eleven translocation)分子在抗海分枝杆菌感染过程中的意义与初步机制。方法SPF级野生型C57BL/6和Tet1基因敲除小鼠(Tet1KO)分别尾静脉接种海分枝杆菌,建立尾静脉感染模型。首先观察比较大体病变如尾巴脓肿形成及面积差异;再用苏木精-伊红染色法染色及透射电镜观察小鼠尾组织病理学变化与差异;免疫组化检测小鼠尾组织中肿瘤坏死因子α(TNF-α)和转化生长因子β(TGF-β)的表达与分布;组织研磨涂抹于7H10固体培养基,计数菌落并统计差异;最后,提取尾组织蛋白,Western blot检测NF-κBp65和TGF-β的表达。结果C57BL/6小鼠感染海分枝杆菌后尾巴上形成明显的脓肿、溃疡等病变;而Tet1KO小鼠病变较轻,仅出现散在的小脓肿,并且尾组织菌载量明显降低。组织病理学观察发现感染海分枝杆菌后,C57BL/6小鼠尾组织中有明显的炎性细胞聚集和浸润,形成肉芽肿样病灶,而Tet1KO小鼠尾组织中无明显炎性细胞浸润;免疫组化检测发现Tet1KO小鼠尾组织中的TNF-α和TGF-β的表达量均比C57BL/6组降低;Western blot检测发现Tet1KO小鼠尾组织中磷酸化NF-κBp65和TGF-β蛋白表达水平显著降低。结论Tet1缺失可以降低海分枝杆菌介导的炎性损伤,且利于宿主对细菌的清除。

Objective To investigate the significance and mechanism of ten-eleven translocation(Tet1)against Mycobacterium marinum(Mm)infection in mice.Methods SPF wild-type C57BL/6 and Tet1-knockout(Tet1KO)mice were injected intravenously with Mm.All mice were monitored and the abscesses formed in tail were observed and quantified.Pathological changes in mouse tail tissues were observed using hematoxylin and eosin(HE)staining and transmission electron microscopy and the differences between the two groups were analyzed.Immunohistochemistry staining was used to detect the expression and distribution of TNF-α and TGF-β in mouse tail tissues.Moreover,mouse tail tissues were cultured on 7H10 plates for bacterial counting.The expression of NF-κBp65 and TGF-β was detected by Western blot.Results Obvious lesions including abscesses and ulcers were formed in the Mm-infected C57BL/6,but only scattered small abscesses were observed in Mm-infected Tet1KO mice.During Mm infection,the bacterial load was gradually increased in C57BL/6 mice,but decreased in Tet1KO mice.Histopathological examination showed that obvious inflammatory cell infiltration and typical granulomatous lesions were found in Mm-infected C57BL/6 mice,while no significant inflammatory cell infiltration was detected in Mm-infected Tet1KO mice.Immunohistochemistry staining demonstrated that the expression of TNF-α and TGF-β was lower in Mm-infected Tet1KO mice than in Mm-infected C57BL/6 mice.Moreover,the expression of phosphorylated NF-κBp65 and TGF-β was significantly reduced in Mm-infected Tet1KO mice as compared with that in Mm-infected C57BL/6 mice.Conclusions Deletion of Tet1 could alleviate the inflammatory damage mediated by Mm and enhance the host immune response to bacteria.