奶牛乳腺炎模型的建立及炎症相关因子基因mRNA转录水平的分析  

Construction of Dairy Cow Mastitis Model and Analysis of mRNA Transcription Level of Inflammation Related Cytokine Genes

作　　者：罗仍卓么 王晋鹏 焦鹏 李彦霞 董益闻 魏大为 王兴平 LUORENG Zhuoma;WANG Jinpeng;JIAO Peng;LI Yanxia;DONG Yiwen;WEI Dawei;WNAG Xingping(School of Agricultural,Ningxia University,Yinchuan 750021,China;Key Laboratory of Ruminant Molecular Cell Breeding,Ningxia Hui Autonomous Region,Yinchuan 750021,China)

机构地区：[1]宁夏大学农学院,银川750021 [2]宁夏回族自治区反刍动物分子细胞育种重点实验室,银川750021

出　　处：《畜牧兽医学报》2022年第8期2763-2772,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(32060749);宁夏高等学校科学研究项目(NGY2020007);宁夏大学引进人才科研启动项目(030900001926)。

摘　　要：旨在探讨金黄色葡萄球菌(S.aureus)型和大肠杆菌(E.coli)型乳腺炎奶牛乳腺组织的炎症相关因子基因的mRNA转录水平。将105 CFU·mL^(-1)的S.aureus和E.coli经乳导管分别注入奶牛乳房,在感染第7天采用活体无菌手术法采集乳腺组织,并采用组织HE染色和免疫荧光法进行乳腺炎模型的鉴定;利用qPCR分别检测了2个诱导组和对照组奶牛乳腺组织的趋化因子家族(CCL2、CCL8、CXCR1、CXCL2和CXCL13)、补体因子(CFI和CFB)、自噬调节因子DEPP1和白细胞介素受体IL21R共9个基因的mRNA转录水平。结果显示:与对照组相比,TLR4/NF-κB炎症相关信号通路中的关键分子(TLR4、NF-κB和TNFα)在2个诱导组乳腺组织中的蛋白表达水平均极显著上调(P<0.01);结合HE染色结果,表明本试验成功构建了2种类型的奶牛乳腺炎活体模型。mRNA转录水平的检测结果表明,与对照组相比,7个基因(CCL2、CCL8、CXCR1、CXCL2、CFI、CFB和IL21R)在2个诱导组乳腺组织中的mRNA转录水平均极显著上调(P<0.001),CXCL13的mRNA转录水平仅在S.aurues诱导组乳腺组织中极显著上调(P<0.01);DEPP1的mRNA转录水平在2个诱导组中均极显著下调(P<0.01)。此外,CCL2、CCL8、CXCR1、CFI和IL21R共5个基因在E.coli诱导组中的mRNA转录水平均极显著高于S.aureus组(P<0.01)。S.aureus和E.coli感染均可导致奶牛产生严重的临床乳腺炎症状,并促使上述炎症相关基因的mRNA转录水平在乳腺组织中发生变化,以应对乳腺炎症的发生与发展过程;趋化因子CCL2等5个基因在E.coli诱导组中的mRNA转录水平显著高于S.aureus诱导组,解释了E.coli常常能引起急性乳腺炎,而S.aureus可引起慢性乳腺炎的原因。上述结果可为深入研究不同类型乳腺炎的分子调控机制提供参考。The study aimed to investigate the mRNA transcription levels of inflammation related cytokine genes in mammary tissues of dairy cows with S. aureus or E. coli type mastitis. S. aureus or E. coli(10~5 CFU·mL) were respectively injected into the cow’s udder through its duct. On the 7 th day of infection, the mammary tissues were collected by aseptic operation in vivo. Then the mastitis models were identified by HE staining and immunofluorescence of mammary tissues. qPCR was used to detect the mRNA transcription levels of nine genes in cow mammary tissues of two induced groups and control group, including chemokine family(CCL2, CCL8, CXCR1, CXCL2 and CXCL13), complement factor(CFI and CFB), autophagy factor DEPP1 and interleukin receptor IL21 R. Compared with the control group, the protein expressions of the key molecules(TLR4, NF-κB and TNF α) in TLR4/NF-κB signaling pathway related inflammation were extremely significantly up-regulated in the mammary tissues of the two induced groups(P<0.01), indicating that two types of mastitis in vivo models were successfully constructed combined with the results of HE staining. The results of mRNA transcription levels detection showed that, compared with the control group, the mRNA transcription levels of seven genes(CCL2, CCL8, CXCR1, CXCL2, CFI, CFB and IL21R) were extremely significantly up-regulated in mammary tissues of the two induced groups(P<0.001), the mRNA transcription levels of CXCL13 were extremely significantly up-regulated only in mammary tissues of the S. aureus induced group(P<0.01), while the expression of DEPP1 was extremely significantly down-regulated in mammary tissues of the both induced groups(P<0.01). In addition, the mRNA transcription levels of five genes including CCL2, CCL8, CXCR1, CFI and IL21R in E. coli induced group were significantly higher than those in S. aureus induced group(P<0.01). S. aureus or E. coli infection can cause serious clinical mastitis symptoms in dairy cows, and promote the abnormal expression of the above inflammation

关 键 词：奶牛 乳腺炎 基因表达 炎症 

分 类 号：S857.26[农业科学—临床兽医学]