Single Cell Related Redox Active Small Molecules In Situ Dynamic Analysis And Research Of Cellular Function

Redox reaction and its balance is one of the most basic and important biochemicalreactions in organisms. For cells, their redox state is determined by oxidizing species(O^2-, H₂O₂, OH, NO, etc) and reducing species （GSH, NADPH, Vitamin C, etc）. Whencells are stimulated by external stimulant or during the process of disease, excessiveexpression of oxidizing species or reducing species will break the cellular redox balance,cause oxidative stress and redox stress and lead to the physiological and pathologicalchanges of cells and organisms. Therefore, it has important significance and applicationvalue in dynamic analysis of redox state in single cells in situ.As we all know, intracellular redox reaction has an important influence in cellproliferation, differentiation, apoptosis, occurrence of major disease and developmentprocess. In order to find real information of individual intracellular redox which iscovered by the traditional bulk cellular analysis, and make a further study of cellularphysiological and pathological process, we did this work. This paper did research indeveloping new technology and new method in dynamic analysis of redox state in singlecells in situ, and did preliminary study on that the redox imbalance associated with cellfunction. This thesis mainly includes the following four parts:In the chapter one, we gave an outline of the biological significance of intracellularredox reaction as well as its dynamic balance, the connection between redox relatedreactive species and redox state, the current research status of redox related reactivespecies. Then, we made a summary of the concept of microfluidic analysis system, itscomposition and its research progress in single cells.In the chapter two, in view of shortage of experimental installations in the analysis ofsingle cell redox state at present, we designed and built a set of microfluidic chip-fluorescence imaging analysis system which can achieve cell feeding, single cellorientation, cultivation, fluorescence labeling and real-time imaging detection ofintracellular redox related reactive species in situ. The system was mainly composed ofgravity-driven component, microfluidic chip, temperature and carbon dioxide controllerand laser scanning confocal microscope. According to the correction of the fluid’s temperature in channel and ROS fluorescence imaging analysis in the two different chips,we selected a more suitable one and investigated the performance of the system. This laid asolid foundation for subsequent experimental application in the dynamic analysis of redoxrelated reactive species in single cells in situ.In the chapter three, in view of the research in mechanism of tumor thermotherapy,cell redox state is mostly aimed at cell extracts or bulk cellular analysis, which could notprovide abundant and real information on tumor thermotherapy. Using microfluidicchip-fluorescence imaging analysis system with reversible near-infrared fluorescentprobe （Cy-O-Eb） which is designed in our laboratory, we studied the changes ofintracellular redox state （GSH/H₂O₂） under thermal induction in single cells. In this study,we achieved dynamic analysis of the changes of intracellular redox state in singleMCF-7/MCF-10A cells under thermal induction and got cell heterogeneity. In addition,we made a preliminarily exploration in the relevance between redox imbalance and cellfunction using flow cytometry, immunofluorescence imaging, western blot etc. Themethod had the advantages of dynamic analysis. This will provide a new method forbiological physiology, pathology relationships, and even have benefit to the early diseasediagnosis and related studies.The fourth chapter is summary and outlook. We designed a set of microfluidicchip-fluorescence imaging analysis system which can achieve cell feeding, single cellorientation, cultivation, fluorescence labeling and dynamic detection of intracellularredox related reactive species in situ. Heat-induced apoptosis of MCF-7is closely relatedto the mitochondrial redox state （GSH/H₂O₂） mbalance. However, If MCF-7mitochondria redox state （GSH/H₂O₂） imbalance is direct induced by ROS and ifcalcium channel is involved in redox state regulation of cell function are for furtherstudy.