本文選題：鼠傷寒沙門菌 + 小鼠�。� 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：沙門菌(Salmonella)是引起人食物中毒的主要病源菌之一。其感染后,能夠通過調(diào)節(jié)宿主細(xì)胞(如巨噬細(xì)胞)功能逃避機(jī)體免疫系統(tǒng)的殺傷,從而在細(xì)胞內(nèi)存活。而在感染早期,機(jī)體也能夠啟動(dòng)天然免疫反應(yīng)(如分泌炎癥因子),以發(fā)揮清除作用。因此,分析沙門菌感染早期炎癥因子的分泌與沙門菌定殖的關(guān)系具有重要意義。本研究首先對(duì)流式蛋白定量分析過程進(jìn)行優(yōu)化,然后選取了野生型鼠傷寒沙門菌和表達(dá)融合蛋白SspH2-EscI的重組鼠傷寒沙門菌為研究對(duì)象,探討其感染早期炎癥因子的分泌與細(xì)菌定殖的相關(guān)性。1鼠源炎癥因子流式蛋白定量試劑盒的優(yōu)化利用流式蛋白定量(cytometric bead array,CBA)技術(shù)分析細(xì)胞因子含量,方法簡(jiǎn)便、易操作且靈敏度高,但試劑成本較高。本實(shí)驗(yàn)以鼠源炎癥因子檢測(cè)試劑盒為參考,分別從降低試劑用量和縮短孵育時(shí)間兩個(gè)方面進(jìn)行優(yōu)化,結(jié)果表明,試劑用量由50μL降至20μL、孵育時(shí)間由2 h縮短至30 min仍然可以得到較好的檢測(cè)效果。以上分析為CBA技術(shù)的推廣應(yīng)用提供了重要數(shù)據(jù)。2利用CBA技術(shù)分析小鼠對(duì)沙門菌感染的應(yīng)答沙門菌感染后,機(jī)體產(chǎn)生應(yīng)答并分泌細(xì)胞因子。本實(shí)驗(yàn)利用CBA技術(shù)動(dòng)態(tài)分析小鼠對(duì)鼠傷寒沙門菌感染(腹腔注射或口服)的應(yīng)答。結(jié)果表明,與未感染組相比,腹腔注射(1×104CFU/只)或口服(2×107CFU/只)感染鼠傷寒沙門菌后,小鼠血液凝固加重;血清中炎癥因子(IFN-γ、IL-6、TNF)的分泌呈現(xiàn)先升后降的趨勢(shì),該變化與脾臟重量、細(xì)菌的體內(nèi)定殖的變化趨勢(shì)一致。腹腔注射和口服途徑感染具有類似的變化趨勢(shì)。這說明炎癥因子的分泌與細(xì)菌的體內(nèi)定殖呈正相關(guān)。3表達(dá)融合蛋白SspH2-EscI的重組沙門菌體外感染巨噬細(xì)胞分析前期研究表明,融合蛋白SspH2-EscI的表達(dá)能夠增強(qiáng)沙門菌誘導(dǎo)的巨噬細(xì)胞內(nèi)炎性體途徑的活化。本實(shí)驗(yàn)以含有空載體的重組菌X4550(pYA3334)和表達(dá)SspH2的重組菌X4550(pYA3334-P-SspH2)為對(duì)照,分析表達(dá)融合蛋白SspH2-EscI的重組沙門菌X4550(pYA3334-P-SspH2-EscI)體外感染RAW264.7細(xì)胞后的細(xì)胞應(yīng)答情況。結(jié)果表明,在感染期間,兩對(duì)照組的變化趨勢(shì)相似。與兩對(duì)照組相比,體外感染(MOI=100)5 h后,X4550(pYA3334-P-SspH2-EscI)感染組細(xì)胞膜受損嚴(yán)重,caspase-1的活化和乳酸脫氫酶的釋放明顯增加,說明細(xì)胞內(nèi)炎性體途徑被激活;另外,細(xì)胞培養(yǎng)上清中炎癥因子的含量在感染期間均呈現(xiàn)下降趨勢(shì),而對(duì)照組明顯不同升高。在細(xì)胞代謝方面,各組在細(xì)胞內(nèi)線粒體膜電位、活性氧、NO和鈣離子濃度等方面的變化趨勢(shì)一致,但在pH方面,X4550(pYA3334-P-SspH2-EscI)組在感染5h后細(xì)胞內(nèi)pH值升高,而對(duì)照組下降。說明炎性體途徑的活化影響了炎癥因子的分泌和細(xì)胞內(nèi)pH值的變化。4表達(dá)融合蛋白SspH2-EscI的重組沙門菌感染小鼠分析據(jù)推測(cè),細(xì)胞內(nèi)炎性體途徑的活化有利于沙門菌的清除。本研究以含有空載體的重組沙門菌X4550(pYA3334)和表達(dá)SspH2蛋白的重組沙門菌X4550(pYA3334-P-SspH2)為對(duì)照,通過感染小鼠(腹腔注射和口服),分析表達(dá)融合蛋白SspH2-EscI的重組沙門菌X4550(pYA3334-P-SspH2-EscI)在小鼠體內(nèi)的定殖能力和機(jī)體炎癥因子分泌情況。結(jié)果表明,在感染期間,兩對(duì)照組呈現(xiàn)相似的變化趨勢(shì),小鼠血液凝固加重,血清中炎癥因子的分泌增加,脾臟重量升高,細(xì)菌定殖嚴(yán)重。而X4550(pYA3334-P-SspH2-EscI)感染組小鼠血液凝固狀態(tài)、脾臟重量、細(xì)菌定殖和炎癥因子分泌均明顯低于對(duì)照組�？诜c腹腔注射感染呈現(xiàn)類似的現(xiàn)象。表明,Ssph2-EscI的表達(dá)降低了沙門菌的體內(nèi)定殖和小鼠血清中炎癥因子的分泌。
[Abstract]:Salmonella (Salmonella) is one of the main pathogenic bacteria causing human food poisoning. After infection, it can escape the killing of the immune system by regulating the function of host cells (such as macrophages) to survive in the cell. In the early stage of infection, the body can also start the natural immune response (such as secreting inflammatory factors) to play a scavenging effect. Therefore, it is of great significance to analyze the relationship between the early inflammatory factors of Salmonella infection and the colonization of Salmonella. Firstly, the quantitative analysis process of convective protein was optimized. Then the wild Salmonella typhimurium and the recombinant Salmonella typhimurium expressing the fusion protein SspH2-EscI were selected as the research object, and the early infection was discussed. The relationship between the secretion of inflammatory factors and bacterial colonization.1 mouse source of inflammatory factor flow protein quantitative reagent box optimization using flow protein quantitative (cytometric bead array, CBA) technology to analyze the cytokine content. The method is simple, easy to operate and highly sensitive, but the reagent cost is high. This experiment took the mouse source inflammatory factor detection kit as the reference. The test was optimized from two aspects: reducing the dosage of reagents and shortening the incubation time. The results showed that the dosage of reagents was reduced from 50 L to 20 mu, and the incubation time was shortened from 2 h to 30 min. The above analysis provided a heavy requirement for the popularization and application of CBA Technology,.2 using CBA technology to analyze the sense of Salmonella in mice. After infected with Salmonella infection, the body produced responses and secreted cytokine. The CBA technique was used to dynamically analyze the response to Salmonella typhimurium infection (intraperitoneal injection or oral) in mice. The results showed that the blood of mice infected with Salmonella typhimurium (1 x 104CFU/) or (2 x 107CFU/) infected with Salmonella typhimurium from the uninfected group The secretion of inflammatory factors (IFN- gamma, IL-6, TNF) in the serum showed a tendency to rise first and then descend, which was in accordance with the tendency of the spleen weight and the colonization of bacteria in the body. Intraperitoneal injection and oral infection showed a similar trend of change. This shows that the secretion of inflammatory factors is positively related to the colonization of bacteria in the body of.3 expression. The preliminary study of recombinant Salmonella infected by recombinant Salmonella protein SspH2-EscI in vitro showed that the expression of fusion protein SspH2-EscI enhanced the activation of Salmonella induced inflammatory pathway in macrophages. The experiment was compared with the recombinant bacteria X4550 (pYA3334) containing empty carriers and the recombinant strain X4550 (pYA3334-P-SspH2) expressing SspH2. The cell response of the recombinant Salmonella X4550 (pYA3334-P-SspH2-EscI) expressing the fusion protein SspH2-EscI in RAW264.7 cells in vitro was analyzed. The results showed that the change trend of the two control groups was similar during the infection period. Compared with the two control group, the cell membrane of the X4550 (pYA3334-P-SspH2-EscI) infection group was seriously damaged after the infection (MOI=100) 5 h. C The activation of aspase-1 and the release of lactate dehydrogenase showed an obvious increase, indicating that the intracellular inflammatory pathway was activated. In addition, the content of inflammatory factors in the cell culture supernatant decreased during the infection, and the control group was significantly different. In cell metabolism, the mitochondrial membrane potential, reactive oxygen species, NO and calcium ions in cells were in cell metabolism. The changes in concentration were consistent, but in pH, X4550 (pYA3334-P-SspH2-EscI) group increased the pH value in the cells after infection with 5h, while the control group decreased. It was suggested that the activation of the inflammatory pathway affects the secretion of inflammatory factors and the changes in the intracellular pH value of the recombinant Salmonella infected mice with the.4 expression of the fusion protein SspH2-EscI. The activation of the intracellular inflammatory pathway is beneficial to the removal of Salmonella. This study uses recombinant Salmonella X4550 (pYA3334) containing empty carriers and recombinant Salmonella X4550 (pYA3334-P-SspH2) expressing SspH2 protein as the control. By infecting mice (intraperitoneal injection and oral), the recombinant Salmonella X4550 (pYA3334-P) expressing the fusion protein SspH2-EscI is analyzed. -SspH2-EscI) the colonization of the mice and the secretion of inflammatory factors in the mice. The results showed that during the infection, the two control groups showed a similar trend, the blood coagulation in the mice increased, the secretion of inflammatory factors in the serum increased, the weight of the spleen increased and the bacterial colonization was severe. The blood coagulation of mice in the X4550 (pYA3334-P-SspH2-EscI) infection group Solid state, spleen weight, bacterial colonization and inflammatory factors were significantly lower than those in the control group. The oral and intraperitoneal infection showed a similar phenomenon. The expression of Ssph2-EscI decreased the colonization of Salmonella and the secretion of inflammatory factors in the serum of mice.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R516.3

【相似文獻(xiàn)】

相關(guān)期刊論文 前2條

1 朱敏;劉正;張國(guó)弛;王蓓;;健康青年人牙菌斑中五種菌屬定殖動(dòng)態(tài)觀察[J];現(xiàn)代口腔醫(yī)學(xué)雜志;1992年03期

2 馬燕;;MRSA去定殖化藥物[J];國(guó)外醫(yī)藥(抗生素分冊(cè));2009年03期

相關(guān)會(huì)議論文 前10條

1 王天龍;汪來發(fā);樸春根;朱天輝;張曉美;;細(xì)菌根部定殖研究進(jìn)展[A];中國(guó)植物病理學(xué)會(huì)2006年學(xué)術(shù)年會(huì)論文集[C];2006年

2 江威;王勇軍;王琦;齊俊生;梅汝鴻;;影響微生物定殖因素的研究[A];中國(guó)植物病理學(xué)會(huì)2006年學(xué)術(shù)年會(huì)論文集[C];2006年

3 李湘民;胡白石;許志剛;Mew,T.W;;拮抗細(xì)菌菌株P(guān)seudomonas fluorescens 7-14在水稻植株上的時(shí)間、空間定殖型[A];2003’華東植物病理學(xué)術(shù)研討會(huì)暨江蘇省植物病理學(xué)會(huì)第十次會(huì)員代表大會(huì)論文集[C];2003年

4 張清霞;吳小剛;張力群;唐文華;;熒光假單胞菌2P24調(diào)控基因突變體定殖能力和生防效果分析[A];中國(guó)植物病理學(xué)會(huì)2007年學(xué)術(shù)年會(huì)論文集[C];2007年

5 張彥;車建美;劉波;唐樂塵;林抗美;;生防菌ANTI-8098A在番茄植株及土壤中定殖特性的研究[A];中國(guó)植物病理學(xué)會(huì)2010年學(xué)術(shù)年會(huì)論文集[C];2010年

6 車建美;劉波;張彥;鄭雪芳;朱育菁;蘇明星;;;青枯病生防菌ANTI-8098A在土壤中定殖特性的研究[A];第三屆全國(guó)微生物資源學(xué)術(shù)暨國(guó)家微生物資源平臺(tái)運(yùn)行服務(wù)研討會(huì)會(huì)議論文摘要集[C];2011年

7 彭于發(fā);張玉勛;黃大f ;張杰;;抗病殺蟲工程菌PT212存活和定殖的初步研究[A];全國(guó)生物防治學(xué)術(shù)討論會(huì)論文摘要集[C];1995年

8 丁海霞;何祥鳳;李燕;王琦;;recA對(duì)Bacillus cereus 905在小麥根部定殖的作用分析[A];中國(guó)植物病理學(xué)會(huì)2012年學(xué)術(shù)年會(huì)論文集[C];2012年

9 楊秀榮;孫淑琴;田濤;;生防細(xì)菌B579在黃瓜根際定殖動(dòng)態(tài)的研究[A];中國(guó)植物病理學(xué)會(huì)2011年學(xué)術(shù)年會(huì)論文集[C];2011年

10 戴楊;米士偉;劉曉光;高克祥;Kurt Mendgen;劉暢;;生防因子球毛殼ND35菌株在植物中的定殖及其分子檢測(cè)[A];中國(guó)植物病理學(xué)會(huì)2011年學(xué)術(shù)年會(huì)論文集[C];2011年

相關(guān)博士學(xué)位論文 前3條

1 馮永君;水稻內(nèi)生優(yōu)勢(shì)成團(tuán)泛菌YS19對(duì)宿主侵染和定殖機(jī)制的研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2001年

2 薛慶云;植物細(xì)菌性青枯病頡頏菌篩選策略研究及生防機(jī)理初探[D];南京農(nóng)業(yè)大學(xué);2008年

3 連玲麗;芽孢桿菌的生防菌株篩選及其抑病機(jī)理[D];福建農(nóng)林大學(xué);2007年

相關(guān)碩士學(xué)位論文 前10條

1 郭麗宏;一些冬蟲夏草定殖真菌分類及其次級(jí)代謝產(chǎn)物的初步研究[D];山西大學(xué);2015年

2 王穎;生防芽孢桿菌ZA1的GFP基因標(biāo)記及其定殖動(dòng)態(tài)研究[D];甘肅農(nóng)業(yè)大學(xué);2015年

3 孫微微;杜仲內(nèi)生真菌的分離鑒定及活性菌株在小麥中的定殖研究[D];安徽農(nóng)業(yè)大學(xué);2014年

4 王tb笙;三種藥劑對(duì)青枯雷爾氏菌在煙草體內(nèi)定殖的影響及機(jī)理研究[D];西南大學(xué);2016年

5 韓俊燕;雙歧桿菌生理特性與其腸道定殖能力相關(guān)性的研究[D];江南大學(xué);2016年

6 韓騰;枯草芽孢桿菌Tpb55菌株對(duì)黑脛病的防治效果、根際定殖規(guī)律及微生態(tài)效應(yīng)[D];中國(guó)農(nóng)業(yè)科學(xué)院;2016年

7 朱丹雪;油茶炭疽病新病原遺傳結(jié)構(gòu)及其內(nèi)生拮抗菌定殖途徑研究[D];中南林業(yè)科技大學(xué);2016年

8 岑浴;多粘類芽孢桿菌在茶葉上的定殖及其對(duì)葉際細(xì)菌群落的影響[D];河北科技大學(xué);2016年

9 曹璞;土壤生態(tài)環(huán)境對(duì)Serratia plymuthica A21-4根際定殖的影響[D];河南農(nóng)業(yè)大學(xué);2009年

10 田濤;蠟樣芽孢桿菌綠色熒光蛋白標(biāo)記及其在小麥上定殖的初探[D];中國(guó)農(nóng)業(yè)大學(xué);2004年

，

本文編號(hào)：1795801