细胞骨架蛋白与汉赛巴尔通体胞内感染机制相关性研究

发布时间：2018-01-01 21:07

本文关键词：细胞骨架蛋白与汉赛巴尔通体胞内感染机制相关性研究　出处：《上海交通大学》2012年博士论文　论文类型：学位论文

【摘要】：汉赛巴尔通体是一类革兰氏染色阴性、营养条件需求苛刻的兼性胞内寄生的需氧小杆菌，主要寄生在血管内皮细胞、上皮细胞及吞噬细胞内，红细胞内寄生是该病原重要感染特点。汉赛巴尔通体是巴尔通体属中传播及致病谱最广泛的一种人畜共患病病原，以猫为天然宿主，通过猫蚤或蜱叮咬在猫群中传播。猫的抓咬可将该病传播给人类，致使感染人群患以局部皮肤损伤及淋巴结肿大为特征的猫抓病，免疫力低下的感染人群则会引起感染性心内膜炎、杆菌样血管瘤、视神经炎、肝脾性紫癜等疾病，对人类的健康带来严重威胁。然而巴尔通体病在全世界范围内仍属于被忽视疾病范畴，该病研究在我国尚处于起步阶段，存在大量误诊漏诊病例，因此在诊断技术、流行病学及致病机制方面亟待深入研究。目前关于宿主细胞与汉赛巴尔通体感染的互作分子筛选鉴定及其与感染机制相关性研究有待进一步阐明。鉴于此，本论文主要从以下几个方面对该病原进行研究：一．CFDA-SE标记示踪汉赛巴尔通体胞内感染研究本研究通过用CFDA-SE标记汉赛巴尔通体，示踪汉赛巴尔通体在细胞内的感染特征。流式细胞术结果证实CFDA-SE染色10min可使汉赛巴尔通体有效标记上绿色荧光，标记的汉赛巴尔通体能正常感染红细胞，在感染复数比(MOI)为1，10和100时感染率分别为4.86%、3.85%和8.47%。标记的汉赛巴尔通体在感染HeLa细胞时，能清晰观察到细菌在胞内形成的菌团结构(invasome)和环核感染的单菌形态。标记的巴尔通体与未标记的巴尔通体在细胞感染力上没有显著差别，说明CFDA-SE标记巴尔通体示踪感染具有较好的可行性。该方法操作简便，避免了抗体标记感染示踪实验费时费力的缺点。同时该方法为实时监测巴尔通体在体内外感染提供了一种技术方法，为本论文后续致病机制的研究奠定基础。二．细胞骨架蛋白与汉赛巴尔通体胞内感染机制相关性研究 ⑴汉赛巴尔通体与宿主细胞互作蛋白的筛选本研究通过生物素标记汉赛巴尔通体外膜蛋白，利用生物素亲和素结合的原理，通过Pull-Down检测技术，筛选能与汉赛巴尔通体相互作用的HeLa细胞膜蛋白，并通过串联质谱对筛选的蛋白条带进行分析。结果证实HeLa细胞中有多种膜蛋白与巴尔通体具有结合互作活性，分子量大小在44-KD与70-KD之间。切取蛋白条带进行串联质谱分析，利用BioworksBrowser3.3软件在HUMAN.v3.87.fasta数据库中检索，，鉴定结果证实互作蛋白主要为细胞骨架蛋白和桥粒相关蛋白。因此，本论文以筛选的细胞骨架中间纤维角蛋白及微丝F-actin肌动蛋白为切入点，进一步研究细胞骨架与汉赛巴尔通体胞内感染的相关性。 ⑵汉赛巴尔通体感染与细胞骨架蛋白表达及重排相关性研究本实验以筛选的目标细胞骨架蛋白基因为靶基因，通过实时荧光定量PCR和western blot方法，从mRNA水平和蛋白水平研究汉赛巴尔通体感染细胞后，细胞骨架相关蛋白表达量的变化。同时运用荧光显微技术及整装细胞电镜技术，观察汉赛巴尔通体感染细胞后细胞骨架的形态变化。结果证实汉赛巴尔通体的入侵能够上调细胞骨架相关蛋白的表达水平，并以细胞骨架中间纤维角蛋白KRT6的表达水平上调最为显著。通过细胞荧光染色证实汉赛巴尔通体感染细胞后，细胞骨架微丝发生重排，形成致密结构分布在感染细菌菌团外周。而角蛋白中间纤维在汉赛巴尔通体感染后发生显著解聚，中间纤维束结构消失。推测角蛋白表达量上调是上皮细胞抗汉赛巴尔通体感染的一种特异性应答。 ⑶细胞骨架蛋白对巴尔通体感染率的影响本实验通过细胞骨架蛋白解聚剂和稳定剂处理细胞，同时用CFDA-SE标记的汉赛巴尔通体感染细胞，通过流式细胞术研究特定细胞骨架蛋白与汉赛巴尔通体感染细胞的相关性。结果证实中间纤维解聚剂(EGTA)处理细胞后，促使汉赛巴尔通体对上皮细胞的入侵。F-actin肌动蛋白解聚剂(细胞松弛素，CB)处理导致细胞微丝解聚，抑制汉赛巴尔通体对细胞的感染，并随着CB浓度的增加对汉赛巴尔通体的入侵抑制效果越明显。细胞经F-actin肌动蛋白固定剂(鬼笔环肽，PHAL)处理后，汉赛巴尔通体感染入侵亦被显著抑制。说明汉赛巴尔通体感染上皮细胞依赖细胞微丝的重排，而细胞中间纤维具有抑制汉赛巴尔通体感染入侵的功能。本研究结果为汉赛巴尔通体对细胞感染机制提供新的解释。
[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.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【相似文献】