In Situ Imaging Analysis Of Mitochondrial DNA And Superoxide Anion Radical In Cells And In Vivo

Liver ischemia-reperfusion(IR)injury occurs during liver transplantation or other liver operations,which can cause cell death and organ failure;and is a common complication in clinical treatment.As the main place for aerobic respiration,mitochondria maintain normal physiological functions of cells and resist IR damage by providing energy to cells.In the process of mitochondrial oxidative phosphorylation for energy supply,mitochondrial DNA(mt DNA)is involved in encoding oxidative phosphorylation-related proteins and provides important support for cells to resist IR damage.It can be seen that the health of mt DNA is closely related to IR damage.Reactive oxygen species(ROS)is an inevitable by-product of mitochondria in the process of oxidative metabolism.Through the highly reactive oxidation reaction,it causes irreversible damage to the protein,DNA,and other molecules in the cell.Among them,the superoxide anion radical(O₂^·-)is the first ROS produced,which will trigger the production of other ROS.The overproduction of O₂^·-may be a key factor leading to mt DNA oxidative damage and ultimately lead to the occurrence of IR damage.Therefore,in order to solve the mystery of the molecular mechanism of IR damage,it is urgent to develop specific imaging analysis tools and establish a high-resolution imaging analysis method for in-situ,real-time analysis of mt DNA and O₂^·-level changes in the process of IR damage.Fluorescence imaging technology has the advantages of high temporal and spatial resolution,in-situ and real-time visualization,and is an effective technical means to solve the above problems.Therefore,this thesis establishes a new method for the in-situ tracing of mt DNA and O₂^·-in the process of IR damage by constructing highly selective and sensitive fluorescent probes.Realize the dynamic imaging analysis of mt DNA and O₂^·-,and further clarify the related signal pathways of IR damage,identify potential therapeutic targets,and screen effective therapeutic drugs.The main content of this article is as follows:1.The fluorescent probe(mt DNA-BP)was constructed of mt DNA and its application in biological imaging.Utilizing the positive charge and size matching of pyridine cations,mt DNA-BP can be accurately positioned in the small grooves of mt DNA.Based on the spatial confinement of DNA,the restricted rotation of the pyridine group reduces the non-radiative energy loss of the excited state of the fluorophore,so that mt DNA-BP emits strong yellow fluorescence and realizes fluorescence-enhanced detection of mt DNA.The experimental results show that mt DNA exhibits the advantages of fast response,high specificity,and high sensitivity.Using mt DNA-BP,we performed in-situ real-time monitoring of mt DNA in the IR process of liver cells by fluorescence imaging,and clarified the signal pathway of ROS-mt DNA-mediated IR damage.In addition,using mt DNA-BP,a method for screening IR damage treatment drugs was established,and pioglitazone and salidroside were found to be effective drugs for IR damage.This work will promote the study of the molecular mechanisms of mt DNA damage-related diseases,and at the same time uses mt DNA as the target molecule provide a drug screening method.2.Fluorescent probe(mt-CA)was constructed and biological imaging application for detecting O₂^·-in mt DNA and mt DNA at the same time.Taking advantage of the characteristics of mitochondrial membrane potential,introducing pyridine cations as mitochondrial targeting groups,the covalently coupled caffeic acid molecule is the recognition group(blue fluorescence)of O₂^·-,and the matching principle of the DNA groove size is used to realize the matching and insertion of the probe into the DNA groove.At the same time,based on the spatial restriction of the DNA groove,the rotation of the pyridine ring of the probe is restricted,so that the entire probe can emit red fluorescence with a longer wavelength.The experimental results show that the probe has the ability to simultaneously monitor the changes of mt DNA and O₂^·-.It provides a powerful tool for exploring the synergistic changes of mt DNA and O₂^·-in the process of IR damage.