牛乳腺炎无乳链球菌β溶血素对奶牛单核巨噬细胞MAPK信号通路调控的分子机制研究

Bovine mastitis Streptococcus agalactiae β hemolysin play a key role in the infection process and which belongs to the non-structural toxicity molecular. Research had showed that human sources of Streptococcus agalactiae β hemolysin promote the macrophage phagocytosis of Streptococcus agalactiae, touch off signal pathway of MAPK and expression of IL-10 and negative regulation of IL-12, NSO2, displayed immune regulation effect in mouse macrophages with sub-lytic concentration. There were differences between bovine and human source strains of β hemolysin gene sequences. It was not clear that the effect of bovine Streptococcus agalactiae β hemolysin regulation of MAPK signal pathway in dairy cows. It had been found that the 100% clinical strains isolated from Inner Mongolia had the β hemolytic phenotype and gene. It was also confirmed that the mutant strain had no survival in macrophages. Based on these, we proposed ideas for to construct β hemolytic gene mutant strain, to test of infection to dairy cows macrophages, to expect the molecular mechanism of β hemolysin regulation of MAPK signaling pathway in dairy cows macrophages. We will to carry on the bovine macrophage internalization assay, cell viability assay, detection of the expression levels of pro-inflammatory cytokines in macrophages and effect of phosphorylation on MAPK signaling pathway protein. This project will explore the molecular mechanism of β hemolysin as signaling molecules regulating innate immunity and will establish the scientific theoretical basis for exploring new ways of prevention and control of bovine mastitis.

牛乳腺炎无乳链球菌β溶血素在致病过程中起到关键作用,属于分泌性非细胞结构型毒力分子。研究表明人源无乳链球菌β溶血素在亚裂解浓度下促使小鼠巨噬细胞对无乳链球菌内化后，通过出触发信号通路MAPK表达IL-10后对IL-12、NSO2负调控出现细胞内“生存”现象，显示免疫调节效应。由于牛源与人源菌株β溶血素基因序列存在较大差异，从而对牛源无乳链球菌β溶血素对奶牛巨噬细胞MAPK信号通路是否具有免疫调控效应并不清楚。已发现内蒙古地区临床菌株100%存在β溶血表型和基因，同时证实β溶血表型突变株无巨噬细胞内“生存”现象。据此提出构建β溶血基因突变株，探索β溶血素对奶牛巨噬细胞MAPK信号通路调控的分子机制的设想。将通过奶牛巨噬细胞内化实验、细胞活力实验，检测巨噬细胞前炎症细胞因子的表达水平及对MAPK信号通路蛋白的磷酸化影响，探索β溶血素作为信号分子调控先天免疫的分子机制，为有效防控提供科学理论。

该项目研究的科学问题是探索明确内蒙古地区牛乳腺炎无乳链球菌溶血素CylE基因对奶牛巨噬细胞完全吞噬功能的免疫调节的分子机制。通过对内蒙古地区奶牛乳腺炎临床乳样中致病性无乳链球菌的分离，鉴定及溶血素CylE基因的克隆分析，构建溶血素CylE基因突变株的构建，奶牛外周血单核巨噬细胞侵染实验，研究结果显示，与巨噬细胞-GBS△cylE组相比，巨噬细胞-GBSwt组中IL-10、TNF-α细胞因子mRNA上调，IL-8、IL-12、NOS2细胞因子mRNA相对转录水平下调，表明无乳链球菌β溶血素CylE基因对细胞因子IL-10及TNF-αmRNA转录水平的影响结果一致，同时对IL-8、IL-12的相对转录水平显示反馈抑制，NOS2 mRNA转录水平显示下调。按照ELISA检测试剂盒说明书检测相关细胞因子蛋白相对表达量，结果显示，与巨噬细胞-GBS△CylE组相比，巨噬细胞-GBSwt组IL-10及TNFα的相对表达量增加，而IL-8、IL-12相对表达量显著降低（P＞1.05和＞0.01）。NOS2的相对表达量降低，与qPCR检测结果相一致。从而明确了内蒙古地区奶牛乳腺炎无乳链球菌溶血素CylE基因对奶牛外周血液单核巨噬细胞的“完全吞噬”功能具有一定的调节机制，无乳链球菌CylE基因通过对奶牛巨噬细胞IL-8、IL-12、NOS2细胞因子的下调机制，促进IL-10及TNFα前炎性细胞因子的上调表达，使得巨噬细胞对无乳链球菌“吞噬”后不能够“完全吞噬”杀死，被吞噬48 h的无乳链球菌仍在巨噬细胞内存在“内生存”现象，这可能是无乳链球菌性牛乳腺炎治疗难度大，常反复的重要分子机制。该研究成果丰富了牛无乳链球菌性乳腺炎致病分子机制的科学理论，为进一步将无乳链球菌溶血素CylE基因做为有效防控的新靶标，研发靶标封闭性抗体及治疗药物奠定了可靠的科学研究基础，对于保障乳源安全具有重要的人口健康战略意义。