Jagau Hilger, Behrens Ina-Kristin, Steinert Michael, Bergmann Simone
Institut für Mikrobiologie, Technische Universität Braunschweig; Devision of Infection Medicine, Department of Clinical Science, Lund University.
Institut für Mikrobiologie, Technische Universität Braunschweig; Medical Microbiology and Hospital Epidemiology, Max von Pettenkofer Institute, Ludwig Maximilians University.
J Vis Exp. 2019 Oct 31(152). doi: 10.3791/60323.
Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells. The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.
肺炎链球菌与内皮细胞表面的相互作用在血流中通过机械敏感蛋白介导，如血管性血友病因子（VWF）。这种糖蛋白会响应剪切应力改变其分子构象，从而暴露出用于广泛宿主-配体相互作用的结合位点。一般来说，已知在确定的剪切流下培养原代内皮细胞可促进特定的细胞分化，并形成类似于血管内衬生理状态的稳定且紧密连接的内皮层。因此，对涉及机械敏感蛋白的细菌病原体与宿主脉管系统之间相互作用的功能分析需要建立能够模拟已知影响血管细胞表面的生理流动力量的泵系统。本研究中使用的微流控装置能够以确定的流速实现流体的连续且无脉冲再循环。计算机控制的气压泵系统通过产生连续、单向且受控的介质流，在内皮细胞表面施加确定的剪切应力。通过使用专为显微镜观察设计的特殊通道载玻片，可以在流动中通过显微镜监测和量化细胞的形态变化以及细菌附着情况。与静态细胞培养感染不同，静态细胞培养感染通常在免疫标记和显微镜分析之前需要进行样品固定，而微流控载玻片在样品固定后既能基于荧光检测蛋白质、细菌和细胞成分；进行连续免疫荧光染色；又能实时进行基于荧光的直接检测。结合荧光细菌和特异性荧光标记抗体，这种感染程序为与血管过程相关的大量科学应用提供了一种高效的多组分可视化系统。
