摘要
为了构建肺炎克雷伯氏菌感染小鼠乳腺炎模型,初步探究小鼠乳腺炎发病的分子机制,试验采用传统细菌培养方法从奶样中分离出致病菌,提取基因组DNA进行PCR快速检测和测序比对。选取12只泌乳Balb/c小鼠,每只小鼠的一侧乳区注射100μL生理盐水作为对照组,在对侧乳区注射100μL浓度为1×109 cfu/mL的肺炎克雷伯氏菌菌液作为感染组。在注射后第1,2,4,8天依次取样,每次3只。采用H.E.染色法观察小鼠乳腺组织的病理变化,实时荧光定量PCR法检测炎症相关基因炎症小体3(NLRP3)、Toll样受体2(TLR2)和Toll样受体4(TLR4)基因的相对表达量。结果表明:试验成功从奶样中分离并鉴定得到肺炎克雷伯氏菌。对照组小鼠第1,2天乳腺腺泡组织结构完整,轮廓清晰,第4,8天乳腺上皮细胞发生溶解并伴有少量脂肪细胞浸润;感染组小鼠的主要病理变化为乳腺上皮细胞之间排列松散,伴有大量脂肪细胞浸润,并且随着感染时间延长小鼠乳腺组织的病理变化越来越明显。感染组小鼠乳腺组织中NLRP3和TLR2基因的相对表达量与对照组相比极显著升高(P<0.01),TLR4基因的相对表达量与对照组相比差异不显著(P>0.05)。说明试验成功构建出肺炎克雷伯氏菌小鼠乳腺炎模型,TLR2、NLRP3基因参与肺炎克雷伯氏菌小鼠乳腺炎的发生,TLR4基因的相对表达量无显著变化。
This study was to establish a mouse model of Klebsiella pneumonia infection, and to explore the molecular mechanism in the pathogenesis of mice mastitis. In this study, pathogenic bacteria were isolated from milk samples by traditional bacterial culture method, and genomic DNA was extracted for rapid PCR detection and sequencing comparison. Twelve lactating female Balb/c mice were injected 100 μL normal saline to one side of each mouse as control group, and 100 μL(1×10~9 cfu/mL) Klebsiella pneumoniae was infected to the other side of each mouse as infection group. Samples were taken successively on day 1, 2, 4 and 8 after treatment, with 3 samples at a time. The pathological changes of mice breast were observed by H.E staining, and the relative expression levels of inflammasome 3(NLRP3), Toll-like receptor 2(TLR2) and Toll-like receptor 4(TLR4) were detected by real-time fluorescence quantitative PCR. The results showed that Klebsiella pneumonia was successfully isolated and identified from milk samples. On day 1 and 2, the mammary gland acini of mice in the control group had complete structure and clear outline, and on day 4 and 8, the mammary epithelial cells(MMEC) lysed with a small amount of adipocyte infiltration. The main pathological symptoms of mice in the infected group were loose arrangement of MMEC with a large number of adipose cells infiltrating, and the pathological changes of mammary gland tissues of mice became more and more obvious with the extension of the infection time. The relative expression levels of NLRP3 and TLR2 in the breast tissue of infected mice were significantly increased(P<0.01), while the relative expression levels of TLR4 were not significantly different from those of the control group(P>0.05). It indicated that the mouse model of mastitis with Klebsiella pneumoniae infection was successfully established. TLR2 and NLRP3 genes were involved in the development of mastitis caused by Klebsiella pneumoniae, there was no significant change in the expression level of TLR4 mRNA.
作者
江孝俊
杨帆
袁露
张嫚玲
李君
蔡亚非
JIANG Xiaojun;YANG Fan;YUAN Lu;ZHANG Manling;LI Jun;CAI Yafei(Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources,College of Life Science,Anhui Nonnal University,Wuhu 241000,China;College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2021年第3期11-15,21,156,共7页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金面上项目(31970413)
国家重点研发计划项目(2018YFC1200201)
安徽省自然科学基金项目(1908085MC83)。
