奶牛金黄色葡萄球菌诱导型临床乳腺炎的病理特征及初步免疫组化研究

发布时间：2018-08-31 10:49

【摘要】：乳腺炎是奶牛场最为常见的疾病之一,主要由微生物感染引起。本研究通过乳头管灌注金黄色葡萄球菌(以下简称金葡菌)方法建立中国荷斯坦牛临床性乳腺炎模型,通过临床检查、BMT检测、SCC检测、组织病理学技术等方法的检测,结果表明成功构建了金葡菌诱导型奶牛乳腺炎模型。在此基础上,通过组织学、免疫印迹、免疫组化染色技术等方法初步研究了金葡菌诱导型乳腺炎乳腺组织PGLYRP1和PTX3蛋白的变化规律,研究了金葡菌诱导型乳腺炎的免疫相关蛋白调控。主要内容和结果如下：1、为建立中国荷斯坦牛诱导型乳腺炎模型,将1×107CFU/mL的金黄色葡萄球菌悬液分别对3头健康且处于泌乳中期奶牛的一个乳区进行乳头管灌注,在攻菌6h、12h、18h、24h里,观察奶牛精神状况、产奶量、食欲、乳汁颜色、体温等,并进行BMT检测和乳中SCC测定,同时进行病理学检查。结果表明：攻菌后奶牛精神萎靡不振、采食量降低、体温升高,攻菌24h时感染乳区颜色变红、肿胀、发热、明显触痛等急性炎症症状,乳样颜色灰白发黄、粘稠,产奶量降低,乳汁与BMT试剂反应后颜色呈绿色或深绿色,摇动时呈胶冻状,部分奶牛乳中SCC高达200万/mL,攻菌24h后试验乳区呈典型的临床型乳腺炎症状,成功地建立了奶牛金黄色葡萄球菌诱导型乳腺炎模型。2、金葡菌诱导性乳腺炎病理特征：HE染色显示金葡菌诱导型乳区其乳腺有明显病变,局部视野可见乳腺间质伴有明显的炎性水肿和增生现象,大量嗜中性白细胞和淋巴细胞浸润。腺泡腔萎缩变形,乳腺上皮细胞连接不紧密,腺泡腔内有少量絮状乳汁和大量分泌物,主要是脱落的乳腺上皮细胞和嗜中性粒细胞、巨噬细胞、淋巴细胞等炎性细胞。透射电镜显示试验乳区乳腺组织中存在大量金葡菌,细菌周围常常有初级溶酶体和次级溶酶体的聚集,乳腺上皮细胞细胞核、线粒体、内质网、溶酶体、细胞膜等均有明显病理现象。细胞核出现不同程度的染色质边集现象,线粒体出现水肿、扩张现象,内质网轻微扩张,网池内出现包容物,细胞质中出现大量空泡,空泡内有糖原、脂滴等内容物。3、Western blotting和免疫组化结果显示：PGLYRP1和PTX3蛋白在对照组和感染组乳腺组织中都有表达,对照组和感染组乳腺组织的蛋白表达量存在明显差异,PGLYRP1和PTX3蛋白在攻菌后蛋白表达量均显著上调。PGLYRP1是分泌性蛋白,主要定位在嗜中性粒细胞细胞膜和细胞质中。PTX3蛋白也是分泌性蛋白,主要定位在巨噬细胞、嗜中性粒细胞的细胞膜或细胞质中。结论：奶牛乳腺炎模型是一种典型、易于建立的乳腺炎动物模型,可以为奶牛乳腺炎的免疫学研究提供更好的试验基础。牛乳腺炎的发生可引起乳腺组织结构的破坏。PGLYRP1和PTX3蛋白可能是奶牛乳腺炎发生的关键蛋白,可作为奶牛乳腺炎抗性研究的候选蛋白。
[Abstract]:Mastitis is one of the most common diseases in dairy, mainly caused by microbial infection. In this study, the clinical mastitis model of Chinese Holstein cattle was established by injecting staphylococcus aureus (Staphylococcus aureus) into the papillary duct. The results showed that the model of cow mastitis induced by Staphylococcus aureus was successfully constructed. On this basis, the changes of PGLYRP1 and PTX3 proteins in breast tissues of Staphylococcus aureus induced mastitis were preliminarily studied by means of histology, immunoblotting and immunohistochemical staining. The regulation of immune-associated protein in induced mastitis of Staphylococcus aureus was studied. The main contents and results were as follows: 1: 1. In order to establish the induced mastitis model of Chinese Holstein cattle, the 1 脳 107CFU/mL Staphylococcus aureus suspension was perfused into the nipple tube of three healthy cows in the middle lactation period. The mental state, milk yield, appetite, milk color, body temperature, BMT, SCC in milk and pathological examination were observed. The results showed that the dairy cows suffered from mental depression, decreased intake, increased body temperature, red, swelling, fever, obvious tenderness and other acute inflammatory symptoms at 24 hours after inoculation. The milk color was gray and yellow, sticky, and milk yield decreased. The color of milk was green or dark green after reaction with BMT reagent, and it was gelatinized when shaking. The SCC in some dairy cows was as high as 2 million / mL. After 24 hours of inoculation, the experimental milk area showed typical clinical mastitis symptoms. A staphylococcus aureus induced mastitis model was successfully established in dairy cattle. The pathological features of staphylococcus aureus induced mastitis were: he staining showed that there were obvious lesions in mammary gland of Staphylococcus aureus induced mastitis. In the local visual field, the stroma of mammary gland was accompanied by inflammatory edema and proliferation, and a large number of neutrophils and lymphocytes were infiltrated. The acinar cavity atrophied and deformed, and the epithelial cells of the mammary gland were not tightly connected. There were a small amount of flocculent milk and a large amount of secretions in the acinar cavity, mainly exfoliated breast epithelial cells and neutrophils, macrophages, lymphocytes and other inflammatory cells. Transmission electron microscopy showed that there were a large number of Staphylococcus aureus in mammary gland tissue in the test area. The primary lysosomes and secondary lysosomes were often gathered around the bacteria, and the epithelial nuclei, mitochondria, endoplasmic reticulum, lysosomes, mitochondria, endoplasmic reticulum, lysosomes of mammary gland, There were obvious pathological phenomena in cell membrane and so on. There were chromatin edge aggregation in nuclei, edema and dilatation of mitochondria, slight dilatation of endoplasmic reticulum, inclusion in reticulum cistern, large number of vacuoles in cytoplasm, and glycogen in vacuoles. The results of Western blotting and immunohistochemistry showed that the protein of 1: PGLYRP1 and PTX3 were expressed in the breast tissues of the control group and the infected group. The expression of PGLYRP1 and PTX3 protein in breast tissues of control group and infected group were significantly up-regulated. PGLYRP1 was secreted protein. PTX3 protein is mainly located in the membrane and cytoplasm of neutrophilic granulocytes. It is also secreted protein and mainly located in the cell membrane or cytoplasm of macrophages and neutrophils. Conclusion: the model of cow mastitis is a typical animal model which is easy to establish and can provide a better experimental basis for the immunological study of dairy cow mastitis. The occurrence of bovine mastitis may cause the destruction of mammary tissue structure. PGLYRP1 and PTX3 proteins may be the key proteins in the development of bovine mastitis and can be used as candidate proteins in the study of cow mastitis resistance.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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