本文選題：乳腺上皮細(xì)胞 + 金黃色葡萄球菌��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：奶牛乳腺炎是危害我國(guó)乃至全世界奶牛業(yè)的一種重大疾病,乳腺炎不僅會(huì)導(dǎo)致牛奶的質(zhì)量及產(chǎn)量下降,而且導(dǎo)致額外勞動(dòng)力以及治療費(fèi)用的增加。金黃色葡萄球菌(Staphylococcus aureus,S.aureus)是引起奶牛乳腺炎的一種重要病原菌,由其引起的奶牛乳腺炎通常表現(xiàn)為持續(xù)性感染、正常奶牛的早期淘汰。同時(shí),由于抗生素的濫用,大部分乳腺炎源金黃色葡萄球菌表現(xiàn)出多重耐藥性,對(duì)公共衛(wèi)生構(gòu)成嚴(yán)重威脅。近年來(lái),人們通過(guò)改善奶牛干奶期管理、調(diào)控營(yíng)養(yǎng)以及制備生物制劑來(lái)預(yù)防奶牛乳腺炎,但效果均不理想。奶牛金黃色葡萄球菌乳腺炎防治困難,根本原因在于現(xiàn)階段人們對(duì)金黃色葡萄球菌感染損傷乳腺機(jī)制還不清楚,嚴(yán)重影響了其防治進(jìn)程。乳腺上皮細(xì)胞是乳腺抵抗病原微生物入侵的第一道防線,病原菌入侵可導(dǎo)致乳腺上皮細(xì)胞的凋亡。NF-κB是具有多向性調(diào)節(jié)作用的蛋白質(zhì)分子,參與調(diào)控多種因子的基因表達(dá),同時(shí),NF-κB是多個(gè)信號(hào)轉(zhuǎn)導(dǎo)通路的交叉點(diǎn),在凋亡中起著非常重要的作用。研究NF-κB如何調(diào)控S.aurues乳腺炎中乳腺上皮細(xì)胞凋亡,探討S.aureus乳腺感染過(guò)程中NF-κB調(diào)控乳腺上皮細(xì)胞凋亡的機(jī)制,有助于更進(jìn)一步理解奶牛S.aureus乳腺感染及轉(zhuǎn)歸機(jī)制,為奶牛乳腺炎防治新型策略奠定理論基礎(chǔ)。本實(shí)驗(yàn)通過(guò)建立體內(nèi)小鼠和體外S.aurues乳腺炎感染模型,通過(guò)測(cè)定感染后小鼠乳腺上皮細(xì)胞(Mouse mammary epithelial cell,MMEC)MMEC的凋亡和NF-κB相關(guān)通路蛋白的轉(zhuǎn)錄和表達(dá),以期闡明NF-κB調(diào)控乳腺上皮細(xì)胞凋亡的機(jī)制,為闡明奶牛S.aureus感染致乳腺損傷的機(jī)理提供理論依據(jù),為尋找干預(yù)乳腺炎癥的新型藥物靶標(biāo)奠定理論基礎(chǔ)。用胰酶消化法分離得到MMEC,將MMEC鋪到6孔板中,待細(xì)胞長(zhǎng)到80%,進(jìn)行分組實(shí)驗(yàn)。體外感染MMEC乳腺炎分為四組,PBS對(duì)照組(NC組)、PDTC處理組(PDTC組)、S.aureus感染組(S.aureus組)、PDTC預(yù)處理S.aureus感染組(PDTC+S.aureus組)。運(yùn)用流式細(xì)胞術(shù)測(cè)定感染后乳腺上皮細(xì)胞凋亡率;Hoechst33342染色對(duì)細(xì)胞凋亡形態(tài)進(jìn)行觀察;利用熒光定量PCR和Western blot檢測(cè)了NF-κB P65和P50以及其IκBɑ的m RNA和蛋白水平,酶聯(lián)免疫法測(cè)定炎性細(xì)胞因子TNF-ɑ和IL-6的分泌量。主要結(jié)果如下:1.1 S.aureus誘導(dǎo)小鼠乳腺上皮細(xì)胞凋亡率收集感染3h后的細(xì)胞,用Annex V和PI雙染法測(cè)定MMEC凋亡率,發(fā)現(xiàn)S.aureus可以引起小鼠乳腺上皮細(xì)胞的凋亡(P0.01),而PDTC+S.aureus組,乳腺上皮的凋亡率比S.aureus感染組顯著下降(P0.01);同時(shí),PBS組與PDTC組沒(méi)有顯著差異。1.2小鼠乳腺上皮細(xì)胞凋亡形態(tài)進(jìn)行觀察細(xì)胞發(fā)生凋亡后,細(xì)胞核皺縮,Hoechst33342染色后,細(xì)胞呈亮藍(lán)色。S.aurues感染組呈現(xiàn)亮藍(lán)色的凋亡細(xì)胞明顯增多,PPDTC+S.aureus組,凋亡的細(xì)胞明顯減少。1.3 Reeal Time PCR檢測(cè)NF-κB P65、P50和IκBɑ轉(zhuǎn)錄水平表達(dá)量對(duì)P65、P50、IκBɑ進(jìn)行轉(zhuǎn)錄水平表達(dá)量的測(cè)定,P65、P50、IκBɑ三個(gè)基因的轉(zhuǎn)錄水平S.aurues感染組跟PBS對(duì)照組相比轉(zhuǎn)錄量增高且差異極顯著(P0.01),而PDTC+S.aureus組,轉(zhuǎn)錄量跟S.aurues感染組相比均下降且差異極顯著(P0.01)。1.4 Western blot檢測(cè)NF-κB P65、IκBɑ、P-P65、P-IκBɑ蛋白水平以β-actin為內(nèi)參,對(duì)P65和IκBɑ蛋白水平和磷酸化水平進(jìn)行測(cè)定,四組中P65蛋白水平?jīng)]有明顯差異,IκBɑ在S.aurues感染組和PDTC+S.aureus組發(fā)生降解,P-P65的蛋白水平在S.aurues感染組和PDTC+S.aureus組表達(dá)量增加,S.aurues感染組P-IκBɑ相較PBS對(duì)照組表達(dá)量增加,PDTC+S.aureus組跟S.aurues感染組比較其表達(dá)量下降。1.5 ELISA檢測(cè)促炎因子TNF-ɑ和IL-6的分泌量S.aurues感染組相較PBS組可顯著提高TNF-ɑ和IL-6的分泌量(P0.05),PDTC+S.aureus組的TNF-ɑ分泌量與S.aurues感染組比較下降顯著(P0.05),IL-6的分泌量與S.aurues感染組比較下降但沒(méi)有顯著差異。用分娩1周后的昆明小鼠進(jìn)行乳腺炎模型的建立,信號(hào)通路抑制劑PDTC使用方式為腹腔注射,實(shí)驗(yàn)分為四組:PBS灌注乳腺組(NC組);PDTC預(yù)處理組(PDTC組);S.aurues感染組;PDTC預(yù)處理后S.aurues感染組(PDTC+S.aureus組)。對(duì)不同處理進(jìn)行全血細(xì)胞計(jì)數(shù);乳腺組織細(xì)菌分離、回收鑒定、計(jì)數(shù);評(píng)估不同處理組的乳腺臨床癥狀變化及病理變化;Tunel檢測(cè)乳腺上皮細(xì)胞凋亡評(píng)價(jià)NF-κB信號(hào)通路對(duì)乳腺炎的影響,結(jié)果如下:2.1不同處理組全血細(xì)胞計(jì)數(shù)對(duì)不同處理組取抗凝血進(jìn)行全血細(xì)胞計(jì)數(shù),血小板及紅細(xì)胞、血紅蛋白、紅細(xì)胞壓積、平均紅細(xì)胞體積、平均紅細(xì)胞血紅蛋白含量、平均紅細(xì)胞血紅蛋白濃度指標(biāo)各處理組之間沒(méi)有差異,S.aurues感染組與PDTC+S.aureus組白細(xì)胞差異顯著(P㩳0.05),S.aureus感染組與PDTC+S.aureus組中性粒細(xì)胞差異顯著,S.aurues感染組與PDTC+S.aureus組淋巴細(xì)胞差異顯著。2.2不同處理組分菌和臨床癥狀變化對(duì)不同處理組S.aureus分菌結(jié)果以LOG10表示,PBS組與PDTC組沒(méi)有分離到細(xì)菌,S.aureus感染組分離到的CFU為8.4±0.05,PDTC+S.aureus組CFU的為6.27±0.048,兩組差異極顯著(P0.01)。PBS和PDTC組小鼠乳腺組織健康呈乳白色,S.aureus感染組和PDTC+S.aureus組均有出血現(xiàn)象,臨床癥狀沒(méi)有差異性。病理組織切片評(píng)分,S.aureus感染組分?jǐn)?shù)為3.4±0.54,PDTC+S.aureus組為2.4±0.54,兩者差異顯著(P0.05)。2.3 Tunel原位凋亡檢測(cè)乳腺組織乳腺上皮細(xì)胞凋亡對(duì)乳腺上皮細(xì)胞進(jìn)行原位凋亡實(shí)驗(yàn),結(jié)果顯示PBS組與PDTC組可見清晰清乳腺腺泡結(jié)構(gòu),凋亡細(xì)胞數(shù)目少,S.aureus感染組乳腺腺泡結(jié)構(gòu)被破壞,有大量的MMEC凋亡,PDTC+S.aureus組凋亡細(xì)胞數(shù)減少。在體內(nèi)動(dòng)物實(shí)驗(yàn)表現(xiàn)了跟體外實(shí)驗(yàn)相似的規(guī)律性。結(jié)果:1乳腺上皮細(xì)胞的凋亡是由金黃色葡萄球菌引起的,信號(hào)通路抑制劑PDTC不能引起乳腺上皮細(xì)胞凋亡作用。NF-κB信號(hào)通路參與了對(duì)乳腺上皮細(xì)胞凋亡的調(diào)節(jié)。2金黃色葡萄球菌引起乳腺上皮細(xì)胞的凋亡是通過(guò)使NF-κB信號(hào)通路及其抑制蛋白IκBɑ的轉(zhuǎn)錄水平升高和P-P65和P-IκBɑ表達(dá)量升高發(fā)揮作用。3 TNF-ɑ和IL-6的分泌量受NF-κB信號(hào)通路的調(diào)控,而IL-6的分泌還可能受其他信號(hào)通路的調(diào)控。4動(dòng)物實(shí)驗(yàn)HE染色,S.aurueus可引起小鼠乳腺炎,PDTC抑制信號(hào)通路后可減輕乳腺炎炎癥發(fā)應(yīng),同時(shí)也可以減少凋亡細(xì)胞數(shù)目,與體外實(shí)驗(yàn)結(jié)果一致。
[Abstract]:Cow mastitis is a major disease that endangers the dairy industry in China and in the world. Mastitis not only leads to the decline in the quality and production of milk, but also leads to the increase of extra labor and treatment costs. Staphylococcus aureus (Staphylococcus aureus, S.aureus) is an important pathogen of cow mastitis. Dairy cow mastitis is usually characterized by persistent infection, early elimination of normal dairy cows. At the same time, the majority of mastitis source of Staphylococcus aureus shows multiple drug resistance due to the abuse of antibiotics, which poses a serious threat to public health. In recent years, people have improved the management of milk cow's dry milk period, regulate nutrition and prepare biological agents in recent years. To prevent cow mastitis, but the effect is not ideal. The prevention and cure of Staphylococcus aureus mastitis is difficult, the basic reason is that the mechanism of the mammary gland infection of Staphylococcus aureus is not clear at the present stage, and it seriously affects its prevention and control process. The invasion of the original bacteria can lead to the apoptosis of the mammary epithelial cells,.NF- kappa B, a protein molecule with multidirectional regulation, and participates in the regulation of gene expression of various factors. At the same time, NF- kappa B is a cross point of multiple signal transduction pathways, and plays a very important role in apoptosis. The study of how NF- kappa B regulates the epithelial cells of the mammary gland in S.aurues mastitis Apoptosis, the mechanism of NF- kappa B regulating the apoptosis of mammary epithelial cells in the process of S.aureus breast infection, will be helpful to further understand the mechanism of mammary gland infection and prognosis of cow S.aureus, and lay a theoretical foundation for the new strategy of cow mastitis prevention and control. This experiment has established the infection model of S.aurues mastitis in vivo and in vitro, through the determination of the sense of the infection. The apoptosis of Mouse mammary epithelial cell (MMEC) MMEC and the transcription and expression of NF- kappa B related pathway proteins in the infected mouse mammary gland in order to elucidate the mechanism of NF- kappa B regulating the apoptosis of mammary epithelial cells, and to provide a theoretical basis for clarifying the mechanism of mammary gland injury induced by S.aureus infection of dairy cows and to find new drugs to interfere with mammary inflammation. The target lay a theoretical basis. MMEC was obtained by trypsin digestion. MMEC was spread to 6 orifice plates, and the cells grew to 80%. The infection of MMEC mastitis in vitro was divided into four groups, PBS control group (NC group), PDTC treatment group (PDTC group), S.aureus infection group (S.aureus group), PDTC pretreated S.aureus infection group (PDTC+S.aureus group). Flow finer was used. The apoptosis rate of the mammary epithelial cells after infection was measured by cytosolic method, the apoptosis morphology was observed by Hoechst33342 staining, and the m RNA and protein levels of NF- kappa B P65 and P50 and its I kappa B were detected by fluorescence quantitative PCR and Western blot. The main results were as follows: 1.1 The apoptosis rate of mouse mammary epithelial cells was induced to collect the cells after 3H infection. The apoptosis rate of MMEC was detected by Annex V and PI double staining. It was found that S.aureus could induce apoptosis of mammary epithelial cells of mice (P0.01), while PDTC+S.aureus group, the apoptosis rate of mammary epithelium was significantly lower than that of the S.aureus infection group (P0.01), while there was no significant difference between the PBS group and the PDTC group. The apoptotic morphology of the mammary epithelial cells of the.1.2 mice was observed. After the cell apoptosis was observed, the nucleus crinkled, and after Hoechst33342 staining, the cell showed bright blue.S.aurues infection group showing bright blue apoptotic cells, and PPDTC+S.aureus group, the apoptotic cells obviously decreased the.1.3 Reeal Time PCR to detect NF- kappa B P65, P50 and transcription level transcription level The transcriptional level of P65, P50, and I kappa B was measured. The transcriptional level of the three genes of P65, P50, I kappa B and the PBS control group increased and the difference was very significant (P0.01). The levels of B P65, I kappa B, P-P65, P-I kappa B were measured with beta -actin as the internal reference. The levels of P65 and I kappa B protein and phosphorylation were measured, and there was no significant difference in the level of P65 protein in the four groups. The expression of P-I kappa B in the urues infection group was higher than that in the PBS control group. The expression of PDTC+S.aureus in the PDTC+S.aureus group and the S.aurues infection group were decreased by.1.5 ELISA, and the secretion quantity of the S.aurues infection group was significantly higher than that in the PBS group. Group comparison decreased significantly (P0.05), the secretion of IL-6 was less than that of S.aurues infection group, but there was no significant difference. The establishment of mastitis model in Kunming mice after 1 weeks of childbirth, the use of signal pathway inhibitor PDTC was intraperitoneal injection, and the experiment was divided into four groups: PBS perfusion Breast Group (NC group); PDTC preconditioning group (PDTC group); S.aurues infection. Group S.aurues infection group (group PDTC+S.aureus) after PDTC pretreatment. The whole blood cell count was carried out in different treatments, the bacterial isolation, recovery identification and counting of breast tissue were used to evaluate the changes of clinical symptoms and pathological changes of breast in different treatment groups, and Tunel was used to evaluate the effects of NF- kappa B signaling pathway on breast epithelial cell withering. The results were as follows: 2.1 the whole blood cell count of different treatment groups took blood cell count, platelet and red blood cell, hemoglobin, hematocrit, mean red blood cell volume, average red blood cell hemoglobin content, and Ping Junhong cell hemoglobin concentration index between different groups, S.aurues infection group and PDTC+S.aure The difference of leucocyte in US group was significant (P? 0.05), the difference of neutrophils between S.aureus infection group and PDTC+S.aureus group was significant. The difference of lymphocyte between S.aurues infection group and PDTC+S.aureus group was significant.2.2 different processing group and clinical symptom changes of S.aureus division results in different treatment groups were expressed LOG10, PBS group and PDTC group did not separate bacteria, S.a The CFU in the UREUS infection group was 8.4 + 0.05, and the CFU in the PDTC+S.aureus group was 6.27 + 0.048. The two groups were extremely significant (P0.01).PBS and PDTC group. The mammary tissues of the mice were milk white, S.aureus infection group and PDTC+S.aureus group had bleeding phenomenon, and the clinical symptoms were not different. The pathological tissue section score and the S.aureus infection group score were 3.4 + 0.. 54, the PDTC+S.aureus group was 2.4 + 0.54, and the difference was significant (P0.05). The apoptosis of mammary gland epithelial cells was detected by.2.3 Tunel in situ apoptosis. The results showed that the structure of the mammary gland was clear in the PBS group and the PDTC group, the number of apoptotic cells was less, and the structure of the mammary gland was destroyed in the S.aureus infection group. A large number of MMEC apoptosis, the number of apoptotic cells in group PDTC+S.aureus decreased. In vivo animal experiments showed similar regularity with in vitro experiments. Results: 1 the apoptosis of mammary epithelial cells was caused by Staphylococcus aureus, and the signal pathway inhibitor PDTC did not cause the apoptosis of mammary epithelial cells by.NF- kappa B signaling pathway involved in the mammary gland The apoptosis of.2 Staphylococcus aureus induced the apoptosis of mammary epithelial cells by making NF- kappa B signaling pathway and its inhibitory protein I kappa B transcriptional level rising and P-P65 and P-I kappa B expression increased to play a role in the regulation of.3 TNF- and IL-6 secretion by NF- kappa signaling pathway. The regulation of signal pathway in.4 animal experiment HE staining, S.aurueus can cause mastitis in mice. After PDTC suppression signal pathway, it can reduce the inflammatory response of mastitis, and also reduce the number of apoptotic cells, which is in accordance with the experimental results in vitro.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.23

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