大肠杆菌及脂多糖对奶牛乳腺上皮细胞基质金属蛋白酶表达的影响  

Effects of E.coli and LPS on Expression of Matrix Metalloproteinases in Bovine Mammary Epithelial Cells

作　　者：张媛媛 丁玉林[1] 刘雅鑫 李晓华 高琼 王凤龙[1] Zhang Yuanyuan;Ding Yulin;Liu Yaxin;Li Xiaohua;Gao Qiong;Wang Fenglong(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia 010018,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018

出　　处：《中国动物检疫》2022年第3期99-110,共12页China Animal Health Inspection

基　　金：国家自然科学基金项目(31460642)。

摘　　要：为研究大肠杆菌(E.coli)及脂多糖(LPS)对奶牛乳腺上皮细胞(BMECs)基质金属蛋白酶(MMPs)表达的影响,以及MMPs与基质金属蛋白酶组织抑制因子(TIMPs)、尿激酶型纤溶酶原激活物(uPA)系统在调控细胞外基质(ECM)代谢中的作用,分别以106 CFU/mL热灭活E.coli菌液、7.5μg/mL LPS以及E.coli菌液与LPS混合液作用于体外培养的BMECs,分别在作用细胞6、12、24、48 h后提取细胞总RNA和总蛋白,通过qPCR、Western blot检测MMP-1、MMP-2、MMP-3、MMP-9、MMP-13、TIMP-1、TIMP-2、uPA、uPAR、PAI-1 mRNA转录水平和相应蛋白表达水平,并通过明胶酶谱试验检测细胞上清液中MMP-2和MMP-9蛋白活性。结果显示:E.coli菌液和LPS作用于BMECs后,MMP-1、MMP-2、MMP-3、MMP-9、MMP-13、TIMP-1、TIMP-2、uPA、uPAR、PAI-1 mRNA转录水平和相应蛋白表达水平均有不同程度上升。MMP-2、TIMP-1、uPA、uPAR、PAI-1 mRNA转录水平在菌液作用后显著上升,MMP-3、MMP-9、MMP-13、TIMP-2、uPAR mRNA转录水平在菌液与LPS联合作用后显著上升;MMP-9、MMP-13、TIMP-2蛋白表达水平在LPS作用后显著上升;TIMP-1、uPA、uPAR蛋白表达水平在菌液作用后显著上升;MMP-9、MMP-13、uPA、uPAR蛋白表达水平在菌液与LPS联合作用后显著上升。3个作用组MMP-2蛋白活性在不同时间均显著上升;菌液、菌液与LPS联合作用12 h时MMP-9蛋白活性显著上升。结果表明,热灭活E.coli菌液和LPS可诱导BMECs中MMP-1、MMP-2、MMP-3、MMP-9、MMP-13、TIMP-1、TIMP-2、uPA、uPAR和PAI-1的表达,并激活uPA系统参与调控MMPs的表达。本试验初步探究了大肠杆菌感染奶牛乳腺组织导致乳腺纤维化的发病机理,为奶牛乳腺炎和乳腺纤维化药物研究提供了依据。In order to study the effects of Escherichia coli(E.coli)on bovine mammary epithelial cells(BMECs)and subsequently on the expression of matrix metalloproteinases(MMPs),and to further clarify the role of MMPs/TIMPs and uPA system in regulating extracellular matrix(ECM)metabolism,106 CFU/mL heat inactivated E.coli solution,7.5μg/mL LPS and combination of E.coli solution and LPS were acted on BMECs that were cultured in vitro respectively.Total RNA and total protein were extracted from the cells after 6,12,24 and 48 hours of treatment,the mRNA transcription levels of MMP-1,MMP-2,MMP-3,MMP-9,MMP-13,TIMP-1,TIMP-2,uPA,uPAR and PAI-1 were detected by qPCR and corresponding protein expression levels were detected by Western blot,and the enzymatic activities of MMP-2 and IMP-9 in cell supernatant were detected by gelatinzymography.The results showed that the mRNA transcription levels of MMP-1,MMP-2,MMP-3,MMP-9,MMP-13,TIMP-1,TIMP-2 and uPAR as well as corresponding protein expression levels increased to different extents after the treatment of E.coli solution and LPS on BMECs.The mRNA transcription levels of TIMP-1 and PAI-1 increased significantly after the treatment of E.coli solution,and those of MMP-3,MMP-13 and TIMP-2 increased significantly after the joint treatment of E.coli solution and LPS;the protein expression levels of MMP-9,MMP-13 and TIMP-2 increased significantly after the treatment of LPS;and those of TIMP-1,uPA and uPAR increased significantly after the treatment of E.coli solution;and the protein expression levels of MMP-9,MMP-13,uPA and uPAR increased significantly after the joint treatment of E.coli solution and LPS.The activity of MMP-2 in the three groups increased significantly at different times;and that of MMP-9 increased significantly after treatment of E.coli solution or joint treatment of E.coli solution and LPS for 12 hours.In conclusion,the expression of MMP-1,MMP-2,MMP-3,MMP-9,MMP-13,TIMP-1,TIMP-2,uPA,uPAR and PAI-1 in BMECs could be induced by E.coli solution and LPS,which could activate uP

关 键 词：大肠杆菌 LPS 乳腺上皮细胞 基质金属蛋白酶 

分 类 号：S852.3[农业科学—基础兽医学]