本文選題：示蹤鼠疫菌 + 吞噬作用　； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文

【摘要】：背景:鼠疫是一種嚴(yán)重威脅人類健康的自然疫源性傳染病,主要經(jīng)蚤叮咬而傳播,亦可經(jīng)呼吸道吸入而感染,傳染性強(qiáng),病死率高。而鼠疫耶爾森氏菌是引起鼠疫的病原體,鼠疫耶爾森氏菌的致病機(jī)制與宿主的免疫反應(yīng)涉及到感染與免疫的兩個(gè)方面。鼠疫菌通過偶然的機(jī)會(huì)(蚤類叮咬或人類剝食受感染的動(dòng)物)進(jìn)入機(jī)體后,首先被局部淋巴組織中的中性粒細(xì)胞和巨噬細(xì)胞吞噬,被中性粒細(xì)胞吞噬的鼠疫菌很快被殺滅,而被巨噬細(xì)胞吞噬的鼠疫菌可以在巨噬細(xì)胞中大量繁殖,并合成大量的毒力因子。當(dāng)巨噬細(xì)胞崩解,鼠疫菌再次從巨噬細(xì)胞中釋放到細(xì)胞外時(shí),便具備了抵抗宿主免疫防御的能力,然后隨淋巴液、血液流經(jīng)其他局部淋巴組織或全身其他臟器中,引起腺鼠疫,肺鼠疫或敗血癥鼠疫等。目前對(duì)鼠疫菌感染引起宿主體液免疫反應(yīng)的研究較深入,并發(fā)現(xiàn)了一些重要的保護(hù)性抗原,如:F1(莢膜抗原)和LcrV(V-抗原)等;然而,越來越多的證據(jù)顯示細(xì)胞免疫在宿主抵抗鼠疫菌的機(jī)制中發(fā)揮了更加重要的作用,例如,將鼠疫菌活化的T細(xì)胞輸入小鼠后能有效保護(hù)吸入性鼠疫菌的感染,也有證據(jù)證實(shí)巨噬細(xì)胞和中性粒細(xì)胞可以有效地吞噬強(qiáng)毒鼠疫菌,但減毒鼠疫菌免疫動(dòng)物后,巨噬細(xì)胞和樹突細(xì)胞吞噬減毒鼠疫菌的能力和抗原提呈的機(jī)制至今尚未闡明。本研究將表達(dá)綠色熒光蛋白的原核表達(dá)質(zhì)粒和表達(dá)紅色熒光蛋白的真核表達(dá)質(zhì)粒導(dǎo)入到鼠疫菌EV76疫苗株和缺失了所有毒力質(zhì)粒的鼠疫菌201株中,構(gòu)建成示蹤鼠疫菌,擬通過觀察巨噬細(xì)胞、中性粒細(xì)胞和樹突細(xì)胞對(duì)它們的吞噬能力和細(xì)胞對(duì)鼠疫菌的殺滅能力,為研究宿主免疫反應(yīng)機(jī)制奠定基礎(chǔ)。 方法:我們將目的片段F1和V以及真核質(zhì)粒pIRES2 DsRed-Express2經(jīng)內(nèi)切酶Bgl II和EcoRI酶切后相連,篩選成功連接,同時(shí)目的片段經(jīng)測(cè)序證實(shí)無突變的單克隆菌落。提取質(zhì)粒后,使用鼠疫菌201株質(zhì)粒缺失株(201P-)和EV76疫苗株作為質(zhì)粒載體,攜帶兩種類型的質(zhì)粒(即表達(dá)綠色熒光蛋白的原核質(zhì)粒pBC-GFP和表達(dá)紅色熒光蛋白的真核質(zhì)粒pIRES2 DsRed-Express2),其表達(dá)不同顏色的熒光蛋白作為鼠疫菌進(jìn)入機(jī)體示蹤的手段,利用磁珠分離技術(shù)從脾細(xì)胞中分離出中性粒細(xì)胞、巨噬細(xì)胞和樹突細(xì)胞,利用流式細(xì)胞儀檢測(cè)細(xì)胞含熒光蛋白的鼠疫菌比例,來觀察三種細(xì)胞吞噬鼠疫菌的情況以及三種細(xì)胞殺滅鼠疫菌的能力等等�；诒緦�(shí)驗(yàn)主要目的是探究吞噬細(xì)胞對(duì)鼠疫菌吞噬能力的觀察,以及吞噬能力是否與毒力相關(guān)等方面,所以我們動(dòng)物實(shí)驗(yàn)只觀察了兩株菌EV76(pIRES2 DsRed-Express2+pBC-GFP)和201P-(pIRES2 DsRed-Express2-F1 +pBC-GFP),而201P-(pIRES2 DsRed-Express2-V+pBC-GFP)只進(jìn)行了體外實(shí)驗(yàn),沒有再進(jìn)行動(dòng)物實(shí)驗(yàn)。我們主要通過兩株菌攜帶不同的質(zhì)粒分別感染兩組小鼠來觀察吞噬細(xì)胞的吞噬情況: EV76(pIRES2 DsRed-Express2+pBC-GFP)和201P-(pIRES2 DsRed-Express2-F1+pBC-GFP),在觀察幾種細(xì)胞吞噬鼠疫菌能力差異的同時(shí),觀察田鼠型鼠疫菌201株在質(zhì)粒完全缺失的情況下作為鼠疫菌DNA疫苗的載體,通過酶聯(lián)免疫法測(cè)定小鼠血清抗體,探討鼠疫菌201P-作為鼠疫菌DNA疫苗載體的可行性。 結(jié)果:本實(shí)驗(yàn)成功將表達(dá)鼠疫菌兩個(gè)重要保護(hù)性抗原的F1和V片段分別克隆到真核表達(dá)質(zhì)粒pIRES2 DsRed-Express2中,經(jīng)過轉(zhuǎn)染293細(xì)胞實(shí)驗(yàn)證實(shí)插入F1和V片段的真核質(zhì)�？梢哉１磉_(dá),將細(xì)胞表達(dá)的融合蛋白從細(xì)胞中釋放出來通過SDS-聚丙烯酰胺凝膠電泳及Western-Blot蛋白免疫活性鑒定,結(jié)果表明融合蛋白F1和V的免疫原性良好,同時(shí)用鼠疫菌感染體外培養(yǎng)的巨噬細(xì)胞RAW,分別將細(xì)胞內(nèi)外的鼠疫菌和細(xì)胞核染色,通過共聚焦顯微鏡成功觀察巨噬細(xì)胞有效地吞噬鼠疫菌,隨后用導(dǎo)入兩種質(zhì)粒的鼠疫菌EV76(pIRES2 DsRed-Express2+pBC-GFP)和201P-(pIRES2 DsRed-Express2-F1+pBC-GFP)免疫小鼠,不同時(shí)間點(diǎn)處死小鼠后,經(jīng)過磁珠分離后的巨噬細(xì)胞,中性粒細(xì)胞,樹突細(xì)胞經(jīng)流式細(xì)胞儀檢測(cè),初步證實(shí)24h分離的中性粒細(xì)胞中殘留有微量的鼠疫菌,而在巨噬細(xì)胞和樹突細(xì)胞內(nèi)鼠疫菌可以存活,并且流式細(xì)胞儀可以檢測(cè)到表達(dá)紅色熒光的巨噬細(xì)胞和樹突細(xì)胞。ELISA檢測(cè)結(jié)果顯示EV76和201P-(含pIRES2 DsRed-Express2-F1)第一次免疫后均有抗體效價(jià),且相差不大;但第二次加強(qiáng)免疫10天后檢測(cè),EV76免疫組抗體滴度明顯高于201P-免疫組,而且這兩次的檢測(cè)結(jié)果經(jīng)統(tǒng)計(jì)學(xué)分析,都具有顯著性差異,具有統(tǒng)計(jì)學(xué)意義。 結(jié)論:我們?cè)O(shè)計(jì)用兩種質(zhì)粒表達(dá)不同的顏色來追蹤鼠疫菌進(jìn)入機(jī)體后與吞噬細(xì)胞的相互作用,鼠疫菌在存活狀態(tài)下可以正常表達(dá)PBC-GFP原核質(zhì)粒表達(dá)綠色熒光蛋白,使我們可以追蹤到鼠疫菌;當(dāng)鼠疫菌被細(xì)胞吞噬并降解掉以后,此時(shí)原核質(zhì)粒在真核環(huán)境中不再表達(dá),而真核質(zhì)粒pIRES2 DsRed-Express2在真核環(huán)境中開始表達(dá)紅色熒光蛋白,有利于我們分析各吞噬細(xì)胞吞噬鼠疫菌的能力。本實(shí)驗(yàn)將雙色質(zhì)粒導(dǎo)入到鼠疫菌201質(zhì)粒缺失突變株和EV76疫苗株后,免疫小鼠,對(duì)鼠疫菌進(jìn)入機(jī)體后吞噬細(xì)胞(包括:中性粒細(xì)胞,巨噬細(xì)胞和樹突細(xì)胞)對(duì)其吞噬能力以及鼠疫菌的存活情況做了初步評(píng)價(jià),在24h中性粒細(xì)胞中檢測(cè)到微量的綠色熒光蛋白,在樹突細(xì)胞和巨噬細(xì)胞中可以檢測(cè)到相對(duì)較多的綠色熒光蛋白,而且EV76株組比201P-組的數(shù)量也相對(duì)多一些。而且在隨后的72h或96h可以檢測(cè)到表達(dá)紅色熒光蛋白的巨噬細(xì)胞和樹突細(xì)胞,而且EV組陽(yáng)性細(xì)胞的數(shù)量也高于201P-組,初步證明不同吞噬細(xì)胞針對(duì)鼠疫菌的吞噬能力差異;鼠疫菌的毒力與吞噬細(xì)胞的吞噬能力有關(guān);同時(shí)我們通過ELISA檢測(cè)兩組小鼠F1抗體的產(chǎn)生情況,通過結(jié)果可以看出201P-組同樣可以產(chǎn)生F1抗體,雖然兩次檢測(cè)抗體滴度均低于EV76組,但可以證實(shí)鼠疫菌201株質(zhì)粒缺失突變株作為DNA疫苗的載體攜帶DNA疫苗的可行性。
[Abstract]:Background: plague is a natural infectious disease, which is a serious threat to human health. It is transmitted mainly by the bite of flea. It can also be infected by inhalation of the respiratory tract. It is highly infectious and with high mortality. The plague Jerson Prand is the pathogen of the plague. The mechanism of the Yersinia pestis's disease and the immune response of the host to the host involves infection and immunity. The two aspect. Yersinia pestis can be devoured by neutrophils and macrophages in the local lymphoid tissue, and the Yersinia pestis, phagocytic by neutrophils, can be killed quickly when the Yersinia pestis enters the body by chance (flea bites or human feeding animals), and the pestis phagocytic bacteria phagocytic by macrophage can be large in macrophages. When the macrophages disintegrate and release them from macrophages to the cells again, it has the ability to resist the immune defense of the host, and then with the lymph, the blood flows through other local lymphatic tissues or other organs of the whole body to cause the plague, the plague of the lung, or the plague of septicemia, and so on. A number of important protective antigens, such as F1 (capsule antigen) and LcrV (V- antigen), are found in the study of the host humoral immune response. However, more and more evidence shows that cellular immunity plays a more important role in the mechanism of host resistance to Yersinia pestis, for example, the T cells that are activated by Yersinia pestis are transported. After entering mice, the infection of Yersinia pestis can be effectively protected. There is also evidence that macrophages and neutrophils can effectively phagocytic Yersinia pestis, but the mechanism of phagocytosis and antigen presentation of Yersinia pestis by phagocytic and dendritic cells has not yet been clarified. The prokaryotic expression plasmid of the fluorescent protein and the eukaryotic expression plasmid expressing the red fluorescent protein were introduced into the 201 strains of Yersinia pestis EV76 vaccine strain and the Yersinia pestis missing all the virulence plasmids. The phagocytosis of the macrophages, neutrophils and dendritic cells and the killing of the cells against Yersinia pestis were to be observed. Inability to lay the foundation for studying the host immune response mechanism.
Methods: We screened the target fragment F1 and V and the true nuclear particle pIRES2 DsRed-Express2 through the endonuclease Bgl II and EcoRI enzyme, and screened the successful connection. At the same time, the target fragment was sequenced to confirm the mutagenless colony. After extracting the plasmid, 201 strains of plasmid deletion strain (201P-) and EV76 vaccine strain of Yersinia pestis were used as plasmid carrier, and two were carried. The plasmids of the species (the prokaryotic pBC-GFP expressing green fluorescent protein and the true nuclear particle pIRES2 DsRed-Express2 expressing the red fluorescent protein), which express the different colors of the fluorescent protein as the means to trace the organism as Yersinia pestis, and separate the neutrophils, macrophages and dendrites from the spleen cells by magnetic bead separation technique. Cells, using flow cytometry to detect the proportion of Yersinia pestis in cells containing fluorescent protein, to observe the situation of phagocytosis of Yersinia pestis in three kinds of cells and the ability of three kinds of cells to kill rodent, etc. based on this experiment, the main purpose of this experiment is to explore the phagocytosis of phagocytic phagocytosis and the relationship of phagocytosis to virulence, etc. So we only observed two strains of bacteria EV76 (pIRES2 DsRed-Express2+pBC-GFP) and 201P- (pIRES2 DsRed-Express2-F1 +pBC-GFP), and 201P- (pIRES2 DsRed-Express2-V+pBC-GFP) only carried out in vitro experiments, and no animal experiments were carried out. We mainly infected two groups of mice by two strains of bacteria carrying different plasmids. Phagocytic phagocytosis: EV76 (pIRES2 DsRed-Express2+pBC-GFP) and 201P- (pIRES2 DsRed-Express2-F1+pBC-GFP). At the same time, observing the difference in the ability of several cells to phagocytic Yersinia pestis, the 201 strains of Yersinia pestis were observed as the carrier of the Yersinia pestis DNA vaccine under the condition of complete deletion of the plasmid, and the mouse blood was determined by enzyme immunoassay. To explore the feasibility of Yersinia pestis 201P- as carrier of Yersinia pestis DNA vaccine.
Results: the F1 and V fragments of two important protective antigens of Yersinia pestis were successfully cloned into the eukaryotic expression plasmid pIRES2 DsRed-Express2. Through the transfection of 293 cells, the true nuclear particles inserted into the F1 and V fragments could be expressed normally, and the fusion proteins expressed by the cells were released from the cells through SDS- polypropylene The immunogenicity of the amide gel electrophoresis and Western-Blot protein showed that the immunogenicity of the fusion protein F1 and V was good. At the same time, the infected macrophage RAW was infected with Yersinia pestis in vitro, and the cells were stained with Yersinia pestis and nuclei respectively. The macrophages were successfully observed by confocal microscopy. EV76 (pIRES2 DsRed-Express2+pBC-GFP) and 201P- (pIRES2 DsRed-Express2-F1+pBC-GFP) in mice were immunized with two plasmids. After the mice were killed at different time points, the macrophages, neutrophils and dendritic cells were detected by the flow cytometry after the magnetic beads were separated at different time points. It was preliminarily confirmed that there were trace residues in the neutrophils separated by 24h. Yersinia pestis can survive in the macrophages and dendritic cells, and the flow cytometry can detect the.ELISA detection results of macrophages and dendritic cells expressing red fluorescence. EV76 and 201P- (including pIRES2 DsRed-Express2-F1) have anti body titer after the first immunization, and the difference is small, but the second time of immunization is 10. The titer of the EV76 immunization group was significantly higher than that of the 201P- immune group, and the results of these two tests were statistically significant and statistically significant.
Conclusion: we designed two kinds of plasmids to express different colors to trace the interaction between Yersinia pestis and phagocyte. Yersinia pestis can express the PBC-GFP prokaryotic plasmid to express green fluorescent protein in the condition of survival, so that we can trace Yersinia pestis; when Yersinia pestis is phagocytic and degraded, it is the original. The nucleolus is no longer expressed in the eukaryotic environment, and the true nucleolus pIRES2 DsRed-Express2 begins to express the red fluorescent protein in the eukaryotic environment, which is beneficial to our analysis of the phagocytic phagocytosis of the phagocytosis. This experiment introduced the double color plasmid into the 201 plasmid missing mutant strain and the EV76 vaccine strain of Yersinia pestis, and immunized mice and Yersinia pestis. The phagocytic cells (including neutrophils, macrophages and dendritic cells) were evaluated for their phagocytosis and the survival of Yersinia pestis after entering the body. A small amount of green fluorescent protein was detected in 24h neutrophils, and relatively more green fluorescent protein could be detected in dendritic cells and macrophages, and EV76 The number of the plant group was also more than that of the 201P- group. Moreover, the macrophages and dendritic cells expressing the red fluorescent protein were detected in the subsequent 72h or 96h, and the number of the positive cells in the group EV was also higher than that in the 201P- group. At the same time, we detected the production of F1 antibody in two groups of mice by ELISA. Through the results, we can see that the 201P- group could also produce F1 antibody, although the titer of the two test antibody was lower than that of the EV76 group, but it could be proved that the 201 strains of the plasmid missing mutant strain of Yersinia pestis were feasible to carry the DNA vaccine as a carrier of DNA vaccine.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378

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7 晨光;食物與免疫力[N];大眾衛(wèi)生報(bào);2001年

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8 莫冰;抗白念珠菌感染免疫實(shí)驗(yàn)研究[D];江西醫(yī)學(xué)院;2001年

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10 顧偉杰;天然抗體3B4的抗金黃色葡萄球菌作用及其機(jī)制研究[D];第四軍醫(yī)大學(xué);2007年

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