【摘要】：乳腺炎是奶牛場最為常見的疾病之一,主要由微生物感染引起。本研究通過乳頭管灌注金黃色葡萄球菌(以下簡稱金葡菌)方法建立中國荷斯坦牛臨床性乳腺炎模型,通過臨床檢查、BMT檢測、SCC檢測、組織病理學技術(shù)等方法的檢測,結(jié)果表明成功構(gòu)建了金葡菌誘導型奶牛乳腺炎模型。在此基礎(chǔ)上,通過組織學、免疫印跡、免疫組化染色技術(shù)等方法初步研究了金葡菌誘導型乳腺炎乳腺組織PGLYRP1和PTX3蛋白的變化規(guī)律,研究了金葡菌誘導型乳腺炎的免疫相關(guān)蛋白調(diào)控。主要內(nèi)容和結(jié)果如下：1、為建立中國荷斯坦牛誘導型乳腺炎模型,將1×107CFU/mL的金黃色葡萄球菌懸液分別對3頭健康且處于泌乳中期奶牛的一個乳區(qū)進行乳頭管灌注,在攻菌6h、12h、18h、24h里,觀察奶牛精神狀況、產(chǎn)奶量、食欲、乳汁顏色、體溫等,并進行BMT檢測和乳中SCC測定,同時進行病理學檢查。結(jié)果表明：攻菌后奶牛精神萎靡不振、采食量降低、體溫升高,攻菌24h時感染乳區(qū)顏色變紅、腫脹、發(fā)熱、明顯觸痛等急性炎癥癥狀,乳樣顏色灰白發(fā)黃、粘稠,產(chǎn)奶量降低,乳汁與BMT試劑反應(yīng)后顏色呈綠色或深綠色,搖動時呈膠凍狀,部分奶牛乳中SCC高達200萬/mL,攻菌24h后試驗乳區(qū)呈典型的臨床型乳腺炎癥狀,成功地建立了奶牛金黃色葡萄球菌誘導型乳腺炎模型。2、金葡菌誘導性乳腺炎病理特征：HE染色顯示金葡菌誘導型乳區(qū)其乳腺有明顯病變,局部視野可見乳腺間質(zhì)伴有明顯的炎性水腫和增生現(xiàn)象,大量嗜中性白細胞和淋巴細胞浸潤。腺泡腔萎縮變形,乳腺上皮細胞連接不緊密,腺泡腔內(nèi)有少量絮狀乳汁和大量分泌物,主要是脫落的乳腺上皮細胞和嗜中性粒細胞、巨噬細胞、淋巴細胞等炎性細胞。透射電鏡顯示試驗乳區(qū)乳腺組織中存在大量金葡菌,細菌周圍常常有初級溶酶體和次級溶酶體的聚集,乳腺上皮細胞細胞核、線粒體、內(nèi)質(zhì)網(wǎng)、溶酶體、細胞膜等均有明顯病理現(xiàn)象。細胞核出現(xiàn)不同程度的染色質(zhì)邊集現(xiàn)象,線粒體出現(xiàn)水腫、擴張現(xiàn)象,內(nèi)質(zhì)網(wǎng)輕微擴張,網(wǎng)池內(nèi)出現(xiàn)包容物,細胞質(zhì)中出現(xiàn)大量空泡,空泡內(nèi)有糖原、脂滴等內(nèi)容物。3、Western blotting和免疫組化結(jié)果顯示：PGLYRP1和PTX3蛋白在對照組和感染組乳腺組織中都有表達,對照組和感染組乳腺組織的蛋白表達量存在明顯差異,PGLYRP1和PTX3蛋白在攻菌后蛋白表達量均顯著上調(diào)。PGLYRP1是分泌性蛋白,主要定位在嗜中性粒細胞細胞膜和細胞質(zhì)中。PTX3蛋白也是分泌性蛋白,主要定位在巨噬細胞、嗜中性粒細胞的細胞膜或細胞質(zhì)中。結(jié)論：奶牛乳腺炎模型是一種典型、易于建立的乳腺炎動物模型,可以為奶牛乳腺炎的免疫學研究提供更好的試驗基礎(chǔ)。牛乳腺炎的發(fā)生可引起乳腺組織結(jié)構(gòu)的破壞。PGLYRP1和PTX3蛋白可能是奶牛乳腺炎發(fā)生的關(guān)鍵蛋白,可作為奶牛乳腺炎抗性研究的候選蛋白。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23

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