本文選題：鼠傷寒沙門菌 + 小鼠　； 參考：《揚州大學》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,能夠通過調節(jié)宿主細胞(如巨噬細胞)功能逃避機體免疫系統(tǒng)的殺傷,從而在細胞內存活。而在感染早期,機體也能夠啟動天然免疫反應(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系具有重要意義。本研究首先對流式蛋白定量分析過程進行優(yōu)化,然后選取了野生型鼠傷寒沙門菌和表達融合蛋白SspH2-EscI的重組鼠傷寒沙門菌為研究對象,探討其感染早期炎癥因子的分泌與細菌定殖的相關性。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)技術分析細胞因子含量,方法簡便、易操作且靈敏度高,但試劑成本較高。本實驗以鼠源炎癥因子檢測試劑盒為參考,分別從降低試劑用量和縮短孵育時間兩個方面進行優(yōu)化,結果表明,試劑用量由50μL降至20μL、孵育時間由2 h縮短至30 min仍然可以得到較好的檢測效果。以上分析為CBA技術的推廣應用提供了重要數(shù)據(jù)。2利用CBA技術分析小鼠對沙門菌感染的應答沙門菌感染后,機體產(chǎn)生應答并分泌細胞因子。本實驗利用CBA技術動態(tài)分析小鼠對鼠傷寒沙門菌感染(腹腔注射或口服)的應答。結果表明,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,該變化與脾臟重量、細菌的體內定殖的變化趨勢一致。腹腔注射和口服途徑感染具有類似的變化趨勢。這說明炎癥因子的分泌與細菌的體內定殖呈正相關。3表達融合蛋白SspH2-EscI的重組沙門菌體外感染巨噬細胞分析前期研究表明,融合蛋白SspH2-EscI的表達能夠增強沙門菌誘導的巨噬細胞內炎性體途徑的活化。本實驗以含有空載體的重組菌X4550(pYA3334)和表達SspH2的重組菌X4550(pYA3334-P-SspH2)為對照,分析表達融合蛋白SspH2-EscI的重組沙門菌X4550(pYA3334-P-SspH2-EscI)體外感染RAW264.7細胞后的細胞應答情況。結果表明,在感染期間,兩對照組的變化趨勢相似。與兩對照組相比,體外感染(MOI=100)5 h后,X4550(pYA3334-P-SspH2-EscI)感染組細胞膜受損嚴重,caspase-1的活化和乳酸脫氫酶的釋放明顯增加,說明細胞內炎性體途徑被激活;另外,細胞培養(yǎng)上清中炎癥因子的含量在感染期間均呈現(xiàn)下降趨勢,而對照組明顯不同升高。在細胞代謝方面,各組在細胞內線粒體膜電位、活性氧、NO和鈣離子濃度等方面的變化趨勢一致,但在pH方面,X4550(pYA3334-P-SspH2-EscI)組在感染5h后細胞內pH值升高,而對照組下降。說明炎性體途徑的活化影響了炎癥因子的分泌和細胞內pH值的變化。4表達融合蛋白SspH2-EscI的重組沙門菌感染小鼠分析據(jù)推測,細胞內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SspH2蛋白的重組沙門菌X4550(pYA3334-P-SspH2)為對照,通過感染小鼠(腹腔注射和口服),分析表達融合蛋白SspH2-EscI的重組沙門菌X4550(pYA3334-P-SspH2-EscI)在小鼠體內的定殖能力和機體炎癥因子分泌情況。結果表明,在感染期間,兩對照組呈現(xiàn)相似的變化趨勢,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,細菌定殖嚴重。而X4550(pYA3334-P-SspH2-EscI)感染組小鼠血液凝固狀態(tài)、脾臟重量、細菌定殖和炎癥因子分泌均明顯低于對照組。口服與腹腔注射感染呈現(xiàn)類似的現(xiàn)象。表明,Ssph2-EscI的表達降低了沙門菌的體內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.

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R516.3

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