| The 21st century is the century of life science.With the continuous deepening of the life science research,it has brought great help to people’s lives,such as improving the quality of life,exploring the mysteries of life,developing new drugs,and helping people to prevent and diagnose diseases.The cell is the basic unit of life.The particularity of cells determines the particularity of organism.understanding the characteristics of cell,we can fundamentally know the life activity process of organisms.Therefore,the cell research has become one of the core research directions of life science.Since its introduction,Scanning Electrochemical Microscopy(SECM)has been widely used in the analysis of biological samples,such as cells,DNA/RNA,enzymes.Compared with other microscopy techniques,SECM has high temporal and spatial resolution.Not only can it obtain the topographic information of the cell surface,but also the electrochemical information of the cell surface.In addition,the electrode probe is not in direct contact with the sample,which avoids cell damage during detection.What’s more,SECM detection needs to be carried out in solution,which can provide cells with an environment similar to that under physiological conditions,so that cells can get a good state in a certain period of time and we can obtain more realistic data.Detecting ROS released by cells to study the properties of cells is one of the important aspects in the field of cell research.However,ROS concentration released by a single cell is low,and the ROS is easy decomposed,which makes in situ detection of ROS difficult.On the other hand,ROS is electrochemically active and suitable for being detected by electrochemical methods.As a good detection method with high temporal and spatial resolution accurately,it has advantages to detect ROS released by a single cell accurately and quantitatively.SECM has micron/submicron-scale electrode probes and a 3D positioning system,which makes it possible to study intercellular signal transduction.Cells do not exist in isolation in organisms,and the life activities of cells depend on the transmission of information between cells.The co-culture system controls the growth of cells.It is suitable to use SECM to study intercellular signal transduction.Detecting the extracellular ROS,p H,K~+ions,surface topography,cell membrane permeability,and cell height can make us understand the state of the cell.To get these data,various instruments are required to test the characteristics of cells together.Different instruments have different requirements for cell sample processing.We can get cell surface topography information,electrochemical information,mechanical information by using different working modes of SECM.It can easily and quickly obtain various information of cells and comprehensively evaluate the state of cells.Finally,the use of SECM combined with the co-culture system and a variety of SECM detection techniques are helpful to research the process of intercellular signal transduction of skin cells under UVB stimulation.This work has potential application value for the study of life processes,drug research,and disease mechanisms.In this thesis,we firstly used the SECM-MPSW to detect in situ ROS and morphology information of single cancer cell in real time.Then,SECM combined with Transwell co-culture system was used to study the intercellular signal transduction between cells under electrical stimulation.In addition,the properties of cells under physiological conditions were examined by using various SECM techniques.Finally,using various SECM techniques combined with Transwell co-culture system,we monitor the intercellular signal transduction between two skin cells after UVB stimulation.The main research work of this thesis is as follows:(1)The level of reactive oxygen species(ROS)is closely related to the balance of intracellular environment and the physiological function of cells.Studying ROS releasing at the single cancer cell level can help us to better study cells from the physiological perspective.Therefore,we designed a new multi-step amperometric scanning method(MPSW)based on scanning electrochemical microscopy(SECM)to detect single extracellular ROS in situ without the interference of dissolved oxygen(DO).Using MPSW method,the electrochemical information of extracellular Fc Me OH,ROS and DO signal molecules can be obtained in a single scanning process.Fc Me OH was used to image cells to observe the morphology of cells.With the elimination of DO interference,the ROS released by cancer cells was qualitatively and quantitatively analyzed.Finally,the release of ROS from cancer cells under different p H conditions was tested,revealing that the release of ROS was different under different p H conditions,and acid conditions could stimulate the release of ROS from cancer cells.This work provides a method that can accurately detect the release of ROS from a single cell without DO interference,and has great application prospects in the field of single cell analysis.(2)In this chapter,we developed a new method of scanning electrochemical microscopy(SECM)combined with Transwell co-culture device,which can be used to study the intercellular signal transduction.In the experiment,SECM potential method was used to detect the extracellular p H,and multi-step amperometric method(MPSW)was used to detect the extracellular ROS.MCF-7 cells were divided into upper and lower layers to culture.The lower layer of MCF-7 cells was signaling cells,and the upper layer of MCF-7 cells was signal-receiving cells.When the signaling cells were stimulated,the extracellular ROS of the signal receiving cells was monitored in situ.It is found that the signal-receiving cells are affected by the signaling cells,and the ROS release will change significantly.When monitoring the change of extracellular p H value in situ,it is found that the H~+ions released by the electrical stimulation signal cells will diffusion and transfer to the upper signal-receiving cells,which will change the amount of ROS released.In addition,we changed the distance between two layers of cells and stimulated different signal cells,and the ROS signal of signal-receiving cells also changed.It is revealed that H~+is one of the signal molecules of intercellular communication.With this SECM experimental device,the process of intercellular signal transduction is revealed,which provides a new method for real-time in situ study of intercellular signal transduction.(3)The skin plays an important role in protecting the human body from environment harm.Understanding the characteristics of skin cells is helpful for studying the mechanism of skin damage,skin health protection and drug development.In this work,scanning electrochemical microscopy(SECM)potential method,multi-step wave amperometry(MPSW),and shear force method were used to detect the changes of extracellular H~+and K~+ions,ROS,and cell height characteristics of HFF and Ha Ca T cells in situ.SECM can be used to detect the changes of various extracellular characteristics in real time and in situ,and analyze the cell characteristics from various aspects to understand the changes of cell activity more comprehensively,laying a foundation for the follow-up study of information transmission between HFF and Ha Ca T skin cells.(4)Skin is the first barrier against the external environment,such as UV radiation,bacteria,viruses,and various harmful substances.UV radiation injury is one of the common skin injuries.UVB refers to the ultraviolet light with the wavelength of 280-320nm.UVB can penetrate the surface skin and have a great impact on Ha Ca T cells and HFF cells.After UVB irradiation on Ha Ca T cells,Ha Ca T cells will release chemical and biological molecules,which will affect the HFF cells.There is intercellular signal transduction between the two cells.In this work,SECM was used to monitor the changes of p He,K~+ions,ROSe,cell permeability,cell height and other characteristics of Ha Ca T and HFF caused by UVB light irradiation in real time and in situ,as well as the signal transduction between the Ha Ca T and HFF cells.This in situ monitoring of skin cell characteristics and changes is helpful to understand the process of signal transduction between skin cells,and further study of the mechanism of skin cell protection,damage and repair,as well as the development of antioxidant drugs. |
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