本文關(guān)鍵詞：細(xì)胞骨架蛋白與漢賽巴爾通體胞內(nèi)感染機制相關(guān)性研究　出處：《上海交通大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 漢賽巴爾通體 細(xì)胞骨架蛋白 感染機制 上皮細(xì)胞

【摘要】：漢賽巴爾通體是一類革蘭氏染色陰性、營養(yǎng)條件需求苛刻的兼性胞內(nèi)寄生的需氧小桿菌，主要寄生在血管內(nèi)皮細(xì)胞、上皮細(xì)胞及吞噬細(xì)胞內(nèi)，紅細(xì)胞內(nèi)寄生是該病原重要感染特點。漢賽巴爾通體是巴爾通體屬中傳播及致病譜最廣泛的一種人畜共患病病原，以貓為天然宿主，通過貓蚤或蜱叮咬在貓群中傳播。貓的抓咬可將該病傳播給人類，致使感染人群患以局部皮膚損傷及淋巴結(jié)腫大為特征的貓抓病，免疫力低下的感染人群則會引起感染性心內(nèi)膜炎、桿菌樣血管瘤、視神經(jīng)炎、肝脾性紫癜等疾病，對人類的健康帶來嚴(yán)重威脅。然而巴爾通體病在全世界范圍內(nèi)仍屬于被忽視疾病范疇，該病研究在我國尚處于起步階段，存在大量誤診漏診病例，因此在診斷技術(shù)、流行病學(xué)及致病機制方面亟待深入研究。目前關(guān)于宿主細(xì)胞與漢賽巴爾通體感染的互作分子篩選鑒定及其與感染機制相關(guān)性研究有待進一步闡明。鑒于此，本論文主要從以下幾個方面對該病原進行研究： 一．CFDA-SE標(biāo)記示蹤漢賽巴爾通體胞內(nèi)感染研究 本研究通過用CFDA-SE標(biāo)記漢賽巴爾通體，示蹤漢賽巴爾通體在細(xì)胞內(nèi)的感染特征。流式細(xì)胞術(shù)結(jié)果證實CFDA-SE染色10min可使?jié)h賽巴爾通體有效標(biāo)記上綠色熒光，標(biāo)記的漢賽巴爾通體能正常感染紅細(xì)胞，在感染復(fù)數(shù)比(MOI)為1，10和100時感染率分別為4.86%、3.85%和8.47%。標(biāo)記的漢賽巴爾通體在感染HeLa細(xì)胞時，能清晰觀察到細(xì)菌在胞內(nèi)形成的菌團結(jié)構(gòu)(invasome)和環(huán)核感染的單菌形態(tài)。標(biāo)記的巴爾通體與未標(biāo)記的巴爾通體在細(xì)胞感染力上沒有顯著差別，說明CFDA-SE標(biāo)記巴爾通體示蹤感染具有較好的可行性。該方法操作簡便，避免了抗體標(biāo)記感染示蹤實驗費時費力的缺點。同時該方法為實時監(jiān)測巴爾通體在體內(nèi)外感染提供了一種技術(shù)方法，為本論文后續(xù)致病機制的研究奠定基礎(chǔ)。 二．細(xì)胞骨架蛋白與漢賽巴爾通體胞內(nèi)感染機制相關(guān)性研究 ⑴漢賽巴爾通體與宿主細(xì)胞互作蛋白的篩選 本研究通過生物素標(biāo)記漢賽巴爾通體外膜蛋白，利用生物素親和素結(jié)合的原理，通過Pull-Down檢測技術(shù)，篩選能與漢賽巴爾通體相互作用的HeLa細(xì)胞膜蛋白，并通過串聯(lián)質(zhì)譜對篩選的蛋白條帶進行分析。結(jié)果證實HeLa細(xì)胞中有多種膜蛋白與巴爾通體具有結(jié)合互作活性，分子量大小在44-KD與70-KD之間。切取蛋白條帶進行串聯(lián)質(zhì)譜分析，利用BioworksBrowser3.3軟件在HUMAN.v3.87.fasta數(shù)據(jù)庫中檢索，，鑒定結(jié)果證實互作蛋白主要為細(xì)胞骨架蛋白和橋粒相關(guān)蛋白。因此，本論文以篩選的細(xì)胞骨架中間纖維角蛋白及微絲F-actin肌動蛋白為切入點，進一步研究細(xì)胞骨架與漢賽巴爾通體胞內(nèi)感染的相關(guān)性。 ⑵漢賽巴爾通體感染與細(xì)胞骨架蛋白表達(dá)及重排相關(guān)性研究 本實驗以篩選的目標(biāo)細(xì)胞骨架蛋白基因為靶基因，通過實時熒光定量PCR和western blot方法，從mRNA水平和蛋白水平研究漢賽巴爾通體感染細(xì)胞后，細(xì)胞骨架相關(guān)蛋白表達(dá)量的變化。同時運用熒光顯微技術(shù)及整裝細(xì)胞電鏡技術(shù)，觀察漢賽巴爾通體感染細(xì)胞后細(xì)胞骨架的形態(tài)變化。結(jié)果證實漢賽巴爾通體的入侵能夠上調(diào)細(xì)胞骨架相關(guān)蛋白的表達(dá)水平，并以細(xì)胞骨架中間纖維角蛋白KRT6的表達(dá)水平上調(diào)最為顯著。通過細(xì)胞熒光染色證實漢賽巴爾通體感染細(xì)胞后，細(xì)胞骨架微絲發(fā)生重排，形成致密結(jié)構(gòu)分布在感染細(xì)菌菌團外周。而角蛋白中間纖維在漢賽巴爾通體感染后發(fā)生顯著解聚，中間纖維束結(jié)構(gòu)消失。推測角蛋白表達(dá)量上調(diào)是上皮細(xì)胞抗?jié)h賽巴爾通體感染的一種特異性應(yīng)答。 ⑶細(xì)胞骨架蛋白對巴爾通體感染率的影響 本實驗通過細(xì)胞骨架蛋白解聚劑和穩(wěn)定劑處理細(xì)胞，同時用CFDA-SE標(biāo)記的漢賽巴爾通體感染細(xì)胞，通過流式細(xì)胞術(shù)研究特定細(xì)胞骨架蛋白與漢賽巴爾通體感染細(xì)胞的相關(guān)性。結(jié)果證實中間纖維解聚劑(EGTA)處理細(xì)胞后，促使?jié)h賽巴爾通體對上皮細(xì)胞的入侵。F-actin肌動蛋白解聚劑(細(xì)胞松弛素，CB)處理導(dǎo)致細(xì)胞微絲解聚，抑制漢賽巴爾通體對細(xì)胞的感染，并隨著CB濃度的增加對漢賽巴爾通體的入侵抑制效果越明顯。細(xì)胞經(jīng)F-actin肌動蛋白固定劑(鬼筆環(huán)肽，PHAL)處理后，漢賽巴爾通體感染入侵亦被顯著抑制。說明漢賽巴爾通體感染上皮細(xì)胞依賴細(xì)胞微絲的重排，而細(xì)胞中間纖維具有抑制漢賽巴爾通體感染入侵的功能。本研究結(jié)果為漢賽巴爾通體對細(xì)胞感染機制提供新的解釋。
[Abstract]:Han Sabar's body is a kind of gram negative, nutritional conditions demanding facultative intracellular aerobic bacteria, mainly parasitic on vascular endothelial cells, epithelial cells and phagocytic cells, red blood cells are the important pathogenic parasitic infection. Han Sabar Barr is the Bartonella spread and pathogenic spectrum of a human and animal the most widely zoonosis pathogen, with cats as natural host, the cat flea or tick bites in the cat. The cat group spread bite can spread the disease to humans, resulting in people suffering from infection to local skin damage and lymph nodes of cat scratch disease, infection immunity will cause coli endocarditis, hemangioma, optic neuritis, purpura and other diseases of liver and spleen, and pose a serious threat to human health. However, the Barr disease in the world still belongs to the category of neglected diseases, the disease The research in China is still in its infancy, there are a lot of misdiagnosis cases in diagnostic techniques, the epidemiology and pathogenic mechanism should be studied thoroughly. At present on the host cell and the Han Sabar infection screening and identification of molecular interactions associated with the infection and its mechanism remains to be clarified. In view of this, this thesis focuses on the study of the pathogen from the following aspects:
A study on the intracellular infection of Han Sabar's common body by a.CFDA-SE marker
The study by Han Sabar was labeled with CFDA-SE, Han Sabar in the tracer infection characteristics in cells. Flow cytometry results confirmed that CFDA-SE 10min can make the green fluorescent staining was effective on Han Sabar mark, mark Han Sabar was normal infected red blood cells, the multiplicity of infection ratio (MOI) for 1,10 and 100 infection rate 4.86%, 3.85% and 8.47%. mark Han Sabar was in HeLa infected cells, can clearly observe the bacteria group structure of bacteria in the form of intracellular loop (invasome) and nuclear infected single bacteria form. Mark Barr on the body and the unmarked Barr was no significant difference in cell infectivity, shows good the feasibility of CFDA-SE labeled Barr tracer infection. This method is simple, to avoid the infection of antibody labeled tracer experiments. At the same time the shortcomings of time-consuming method for real-time monitoring of Barr It provides a technical method for the infection in the body and outside the body, which lays the foundation for the subsequent research on the pathogenesis of this thesis.
Two. A study on the relationship between cytoskeleton and the mechanism of the intracellular infection of Han Sabar's body
The Bartonella henselae host cells and screening the protein interaction
This study by using a biotin labeled Han Sabar body outer membrane protein, avidin biotin binding principle used by Pull-Down technology and HeLa cell membrane protein screening and Han Sabar body interaction, and through tandem mass spectrometry on protein screening with analysis. The results demonstrated that HeLa cells have a variety of membrane proteins and with Bartonella with the interaction of activity, molecular weight between 44-KD and 70-KD. The protein bands were cut from the tandem mass spectrometry analysis, retrieval in HUMAN.v3.87.fasta database by using BioworksBrowser3.3 software, the identification result confirmed that protein interaction for cytoskeletal proteins and desmosome related proteins. Therefore, the screening of keratin intermediate filament cytoskeleton F-actin and actin microfilaments as the starting point, further Study on the correlation between the cytoskeleton and the Han Sabar intracellular infection.
The relationship between Bartonella henselae infection and cell skeleton protein expression and rearrangement
In this experiment, the target of cytoskeletal protein gene screening for target gene by real-time quantitative PCR and Western blot, from the mRNA level and protein level of Han Sabar quintana infected cells, the expression of cytoskeleton associated protein. At the same time using fluorescence microscopy and whole cell electron microscope to observe the morphological changes of the cytoskeleton of Han Sabar after infection of cells. The results show that the Han Sabar invasion of Bartonella can up regulate the expression of cytoskeleton related proteins, and to the intermediate filament cytoskeleton protein KRT6 expression level increased most significantly. The cell fluorescence staining confirmed that Han Sabar was infected cells, microfilament cytoskeleton rearrangement, forming a dense structure distribution in bacterial infection group peripheral keratin intermediate filament. While significant depolymerization in Han Sabar body after infection, the intermediate fiber The beam structure disappears. The up-regulated expression of keratin is a specific response to the anti Han Sabar body infection of epithelial cells.
The effects of cytoskeletal protein against Bartonella infection rate
This experiment through the cytoskeleton depolymerization agent and stabilizer of cells labeled with CFDA-SE, while Han Sabar was infected cells, the correlation of flow cytometry to study specific cytoskeletal proteins and the Han Sabar infected cells. The results show that the intermediate filament depolymerizing agent (EGTA) in cells treated by the invasion prompted Han Sabar.F-actin actin depolymerizing agent on epithelial cells (cytochalasin, CB) treatment resulted in actin depolymerization, inhibit the body of Han Sabar cell infection, and with the increase of CB concentration on the inhibitory effect of the Han Sabar invasion is more obvious. The cells with F-actin actin fixing agent (phalloidin, PHAL) after the treatment, Han Sabar quintana was also significantly inhibited. The invasion of infection that depends on the rearrangement of microfilament Han Sabar quintana infection of epithelial cells, and intermediate fiber cells to inhibit Hansell The results of this study provide a new explanation for the mechanism of cell infection in Han Sabar's body.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

【相似文獻】

相關(guān)期刊論文 前10條

1 佟世德;巴爾通體及其相關(guān)疫病[J];華南國防醫(yī)學(xué)雜志;2003年03期

2 栗冬梅;劉起勇;俞東征;張麗云;董興齊;;蚤、蜱中巴爾通體的分離培養(yǎng)及檢測鑒定[J];中國人獸共患病雜志;2005年12期

3 楊小冉;劉起勇;;漢賽巴爾通體的研究進展[J];中國媒介生物學(xué)及控制雜志;2005年06期

4 楊慧;白鶴鳴;楊發(fā)蓮;;巴爾通體菌株的保存與復(fù)蘇[J];地方病通報;2007年02期

5 楊發(fā)蓮;白鶴鳴;楊慧;;云南巴爾通體研究概況[J];醫(yī)學(xué)動物防制;2008年05期

6 栗冬梅;張建中;劉起勇;;中國巴爾通體與相關(guān)疾病的研究進展[J];中國人獸共患病學(xué)報;2008年08期

7 楊發(fā)蓮;巴爾通體在云南省的調(diào)查研究[J];地方病通報;2005年01期

8 白鶴鳴;楊慧;楊發(fā)蓮;;巴爾通體致病機理研究進展[J];中國人獸共患病學(xué)報;2006年12期

9 楊發(fā)蓮;白鶴鳴;楊慧;;云南巴爾通體的系統(tǒng)發(fā)育多樣性分析[J];中國熱帶醫(yī)學(xué);2008年12期

10 劉小閃;劉起勇;;漢賽巴爾通體媒介和宿主的實驗室研究進展[J];中國媒介生物學(xué)及控制雜志;2006年02期

相關(guān)會議論文 前4條

1 陳永金;;巴爾通體[A];第二屆浙江中西部科技論壇論文集第七卷（預(yù)防醫(yī)學(xué)分卷）[C];2005年

2 陳永金;;巴爾通體(Bartonalla)[A];應(yīng)對突發(fā)公共衛(wèi)生事件論壇論文集[C];2005年

3 程王琨;李玉峰;丁維民;劉朋;;漢賽巴爾通體17-kDa蛋白的表達(dá)及間接ELISA方法的建立[A];中國畜牧獸醫(yī)學(xué)會家畜傳染病學(xué)分會第八屆全國會員代表大會暨第十五次學(xué)術(shù)研討會論文集[C];2013年

4 李志芳;劉起勇;張衛(wèi)東;宋秀平;栗冬梅;馬懷雷;趙帆;孟鳳霞;任東升;靳建超;林杰;吳海霞;;長角血蜱感染漢賽巴爾通體后血淋巴超氧化物歧化酶變化的初步研究[A];中國衛(wèi)生有害生物防制協(xié)會2012年年會論文匯編[C];2012年

相關(guān)博士學(xué)位論文 前3條

1 朱彩霞;細(xì)胞骨架蛋白與漢賽巴爾通體胞內(nèi)感染機制相關(guān)性研究[D];上海交通大學(xué);2012年

2 栗冬梅;漢賽巴爾通體外膜蛋白質(zhì)組學(xué)研究[D];中國疾病預(yù)防控制中心;2009年

3 趙帆;中國漢賽巴爾通體的分子分型研究[D];中國疾病預(yù)防控制中心;2011年

相關(guān)碩士學(xué)位論文 前7條

1 許傳彬;脈沖場凝膠電泳技術(shù)在巴爾通體的建立及在流行病學(xué)中的應(yīng)用[D];中國疾病預(yù)防控制中心;2008年

2 王逸曉;中國部分地區(qū)貓群中漢塞巴爾通氏體分子流行病學(xué)調(diào)查研究[D];上海交通大學(xué);2011年

3 俞向前;浦東地區(qū)貓群漢賽巴爾通體流行病學(xué)調(diào)查[D];上海交通大學(xué);2009年

4 武燕斌;漢賽巴爾通體P17基因原核表達(dá)及間接ELISA檢測方法的建立[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2011年

5 李晶晶;漢賽巴爾通體體外紅細(xì)胞感染實驗[D];上海交通大學(xué);2011年

6 苗志剛;巴爾通體脂肪酸分類鑒定技術(shù)研究[D];山東大學(xué);2012年

7 王君;漢賽巴爾通體重組外膜蛋白Pap31的研制和抗原性分析[D];汕頭大學(xué);2008年

本文編號：1366199