Regulation Of Extracellular Microenvironment And Investigation Of Vaccinia Virus-induced Cell Migration On Microfluidic Chip

Virus-induced cell migration is closely related to the activation of immune system in human body,vascular diseases occurring and tumors formation.Thus,it is significant to investigate the cell migration behaviors induced by virus in vitro for revealing the mechanism of related physiological or pathological processes.Vaccinia virus(VACV)is a large DNA virus belonging to the poxvirus family.It was widely known as a vaccine against smallpox.In recent years,studies have found that vaccinia virus could induce cells to migrate,and speculated that the occurrence of migration behavior may be related to the rapid cell-to-cell spread mechanism of VACV.So far,limited studies involving cell migration induced by VACV are based on the traditional cell culture techniques in vitro,and the extracellular microenvironment has rarely been taken into account.In vivo,cells are innately exposed to complex microenvironments,and highly integrated cell migration process can be regulated by a series of external cues.Therefore,it has great significance to explore the influence of extracellular microenvironment on the cell migration induced by VACV for comprehensively and deeply understanding the migration behaviors of infected cells in vivo,revealing the regulation mechanism of external cues on VACV-induced cell migration,and elucidating the relationship between cell migration and virus dissemination.As an emerging technology,microfluidic chip has been widely used in the field of cell biology due to the characteristics of low sample consumption,convenient integration and high throughput.Compared with traditional biological methods,microfluidic chip platform has obvious advantage in flexibly controlling cells and accurately simulating the extracellular microenvironment.On microfluidic chip,it is easy to achieve stable concentration gradient of chemical factor,different kinds of fluid shear stress,and various topography structures,which provides a promising platform to study the effect of various biophysical or biochemical factors naturally existing in body on the cell migration in vitro.The establishment of representative biological models probably facilitates the discovery of new phenomenon and mechanism of biology.Based on the above background,in this dissertation,we are focused on the regulation of extracellular microenvironment on microfluidic chip and VACV-induced cell migration to carry out the following research works:1.We developed a novel microfluidic-based multi-shear cell migration assay platform for the study of the effect of shear stress on cell migration induced by VACV.Regular wound areas with different width were formed by microvalves,which could effectively avoid cell damage in traditional scratch assay.Meanwhile,utilizing this special channel structure,multi-shear stresses covering a wide range were easily obtained by simply changing the inlet velocity.This promising platform integrating improved method for wound areas formation with multi-shear stresses has a potential to explore mechanical stimuli on cell migration.Using this novel platform,we found that infected cells were more elongated and tended to migrate along the flow direction.Shear stress enhanced the natural directional persistence and accelerated the velocity of infected cell migration.Further study found that reduced peripheral lamellae and confined axial lamella to flow direction were responsible for the increased directionality of cell migration under shear stress.Golgi complex reoriented and relocated behind the nucleus and aligned to the flow direction in infected migratory cells,which provides important information to explore the mechanism of Golgi complex reorientation in migratory cells.This work fills the blank of external regulators influence on the VACV-induced cell migration and opens a window for VACV-induced cell migration in vitro.2.We designed an open microfluidic chip based on the microgrooved substrate to explore the influence of topography on VACV-induced cell migration.The microgrooved PDMS membrane simulated the topographical structure of extracellular matrix to some extent.Moreover,the open microfluidic chip easily combined with the traditional biology technology enabled to subsequently analyze the expression of related proteins in the migration process and reveal the molecular mechanism.On this platform,we found that topography of substrate has extensive influence on the migratory behaviors induced by VACV from velocity and directionality of migration to subcellular structures including lamellae formation and Golgi complex relocation.We suggested that microgroove substrate influenced the lamellae formation in migratory cells by regulating the F-actin rearrangement through contact guiding mechanism,which promoted the directional cell migration induced by VACV.Further study found that microgroove substrate could induce migratory cells to form long extended protrusions containing a large number of virus particles,which was beneficial to the rapid and directional cell-to-cell spread of VACV.This work provides sufficient information for comprehensively and deeply understanding the infected cells exposed to ECM migration behaviors.The precisely controlling of the directional cell migration induced by VACV lays the foundation for revealing the relationship between virus spread and virus-induced cell migration.3.We developed a cell co-culture microfluidic chip integrated with the confined microchannels to investigate the effect of confinement on the VACV-induced cell migration and the influence of cell migration on virus cell-to-cell spread.Confined microchannels simulated the three-dimensional restrictive growth environment of cells in vivo in some extend.Cells infected with VACV exhibited accelerated and directional enhanced migration behaviors in confined microchannels.Meanwhile,confined microchannels have influence on the subcellular structure of migratory cells,including the formation of lamellae and relocation of Golgi complex.On this platform,we realized the uninfected and infected cells pattern by hydraulic cell loading mode and microchannels height restriction.We firstly observed that directional cell migration induced by VACV in confined microchannels triggered the long-range cell-to-cell spread of VACV by directed contacted with normal cells.Normal cells infected by migratory cells could also migrate.The process that migration induces infection,and infection triggers migration,probably facilitates the rapid cell-to-cell spread of VACV.Our work provides important information for revealing the mechanism of rapid cell-to-cell spread of VACV,and also provides new method and new ideas for studying the relationship between virus-induced cell migration and virus dissemination.