机构地区:[1]内蒙古民族大学生命科学与食品学院,内蒙古通辽028043 [2]内蒙古自治区乳源性致病菌防控工程技术研究中心,内蒙古通辽028043 [3]内蒙古民族大学乳源性致病菌研究所,内蒙古通辽028043
出 处:《内蒙古民族大学学报(自然科学版)》2024年第6期90-96,共7页Journal of Inner Mongolia Minzu University:Natural Sciences Edition
基 金:国家自然科学基金项目(32060798);内蒙古自治区科技计划项目(2021GG0001);内蒙古自治区自然科学基金项目(2021MS03084)。
摘 要:为研究牛乳腺炎粪肠球菌β溶血素cylA基因对牛单核巨噬细胞(BMC-7)吞噬功能和免疫调节的分子机制,利用牛乳腺炎粪肠球菌临床分离的野生型菌株(BME1708)及cylA基因缺失突变株(BME1708△cylA)对牛巨噬细胞进行侵染,利用MTT检测法检测细胞活性;在明确细胞活性不受影响的条件下,提取被侵染的BMC-7总mRNA,利用RT-qPCR检测细胞因子IL-4、IL-8、IL-10、IL-12、TNF-α以及NOS2的转录水平差异,采用ELISA法检测相应表达水平;利用Western blot检测牛乳腺炎粪肠球菌cylA基因对BMC-7的p38 MAPK信号通路磷酸化水平的影响;利用p38 MAPK信号通路抑制剂(PD 169316)孵育BMC-7,ELISA法检测相应细胞因子表达水平,以明确cylA基因调控该信号通路的分子机制。结果显示:与BME1708组相比,BME1708△cylA组在IL-10和TNF-α转录水平下调,IL-4、IL-8、IL-12以及NOS2转录水平上调,差异均具有统计学意义(P<0.05);与BME1708组相比,BME1708△cylA组IL-10表达水平下调,IL-4、IL-8、IL-12表达水平上调,差异均具有统计学意义(P<0.05);TNF-α的表达水平降低,但是差异不显著;对p38 MAPK磷酸化检测结果显示,粪肠球菌cylA基因可以激活p38 MAPK信号通路并使其磷酸化;在PD 169316孵育后,与未添加抑制剂组相比,添加抑制剂组的IL-10、TNF-α的表达水平下调,IL-4、IL-8、IL-12的表达水平上调,差异均具有统计学意义(P<0.05)。上述结果表明,p38 MAPK信号通路为粪肠球菌β溶血素cylA基因发挥免疫调节机制的重要通路,该结论为奶牛乳腺炎的治疗及防控提供新的靶位,可通过其毒力的钝化以及对p38 MAPK信号通路进行识别及信号通路封闭,对粪肠球菌感染进行有效防控。This experiment is to study the bovine mastitis and Enterococcus faecalis the molecular mechanism ofβhemolysin cylA gene on phagocytic function and immune regulation of bovine monocyte macrophages BMC-7.BMC-7 were infected by wild-type BME1708 strains and cylA gene deletion mutants BME1708△cylA of clinical isolates of bovine mastitis Enterococcus faecalis,and the cell activity was detected by MTT assay;Under the condition of ensuring that cell activity was not affected,the total mRNA of infected BMC-7 was extracted,and the transcription levels of cytokines IL-4,IL-8,IL-10,IL-12,TNF-αand NOS2 were detected by RT-qPCR,and the corresponding expression levels were detected by ELISA;Western blot was used to detect the effect of cylA gene of Enterococcus faecalis with bovine mastitis on the phosphorylation level of p38 MAPK signal pathway of BMC-7;Mononuclear macrophages were incubated with p38 MAPK signaling pathway inhibitors PD 169316,and the corresponding cytokine expression levels were detected by ELISA to clarify the molecular mechanism by which the cylA gene regulates this signaling pathway.Results display:Compared with the BME1708 group,the transcription levels of IL-10 and TNF-αwere down-regulated in BME1708△cylA group,while the transcription levels of IL-4,IL-8,IL-12,and NOS2 were up-egulated,with statistical significance(P<0.05);Compared with the BME1708 group,the transcription levels of IL-10 was down-regulated in BME1708△cylA group,while the expression levels of IL-4,IL-8,IL-12 were up-regulated,with statistical significance(P<0.05).The expression level of TNF-αdecreased,but the difference was not statistically significant;The p38 MAPK phosphorylation test showed that the p38 MAPK signaling pathway could be activated and phosphorylated by the cylA gene of Enterococcus faecalis;After incubation with PD 169316,compared with the non inhibitor group,the IL-10 and TNF-αlevels in the inhibitor added group were down-regulated.The expression levels of IL-4,IL-8,and IL-12 were up-regulated,and the differ
关 键 词:牛乳腺炎 粪肠球菌 溶血素cylA基因 细胞因子 p38 MAPK
分 类 号:TS254.58[轻工技术与工程—水产品加工及贮藏工程]
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