| CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEWED | 第1-64页 |
| PARTⅠ: Usage and research of thrombolytic therapy | 第17-19页 |
| ·Current of thrombolytic therapy | 第17-18页 |
| ·Future Directions of Thrombolytic Therapy | 第18-19页 |
| PART Ⅱ: Main fibrinolytic enzymes | 第19-28页 |
| ·Coagulation and fibrinolytic system | 第19-21页 |
| ·Coagulation system | 第19页 |
| ·Fibrinolytic system | 第19-21页 |
| ·Main fibrinolytic agents | 第21-28页 |
| ·Streptokinase (SK) | 第21-22页 |
| ·Urokinase (UK) | 第22页 |
| ·Tissue-type plasminogen activator (t-PA) | 第22-23页 |
| ·Staphylokinase (SAK) | 第23页 |
| ·Nattokinase (NK) | 第23-28页 |
| PART Ⅲ: Targeted thrombolysis; third-generation thrombolytic drugs | 第28-45页 |
| ·Antibody-targeting technique | 第29-37页 |
| ·Antibody targeted thrombolytic agents | 第29-32页 |
| ·Phage display antibody libraries | 第32-37页 |
| ·Application of recombinant adenovirus in vascular diseases management | 第37-43页 |
| ·Gene therapy and applications | 第37-38页 |
| ·Adenovirus vectors | 第38-40页 |
| ·A New adenovirus vector system, AdEasy system | 第40页 |
| ·Adenovirus vectors usage in cardiovascular gene therapy | 第40-43页 |
| ·New report gene, gfp | 第43-45页 |
| PART Ⅳ: Vascular endothelial cells and oxidative injury | 第45-55页 |
| ·Free radical and its toxicities | 第45-46页 |
| ·Effects of anti-oxidants on the vascular diseases | 第46-47页 |
| ·The action of hemoproteins in vascular diseases | 第47-48页 |
| ·Endothelial cell injury by oxidation stress | 第48-52页 |
| ·Endothelial cell apoptosis | 第52-55页 |
| PART Ⅴ: Bacteria plasminogen activator and infection | 第55-63页 |
| ·Plasminogen and plasmin | 第55-56页 |
| ·The relationship between bacterial PAs and its infection | 第56-60页 |
| ·E. coli periplasmic proteases DegS is associated with infection | 第60-63页 |
| PART Ⅵ: Purpose of this research (中文) | 第63-64页 |
| CHAPTER TWO: GENERAL MOLECULAR TECHNIQUES | 第64-88页 |
| ·Procedures for extraction of plasmid DNA | 第64-66页 |
| ·Alkaline method (small scale extraction) | 第64-65页 |
| ·Alkaline method (large scale extraction) | 第65-66页 |
| ·Digestion, recovering and ligation of DNAs | 第66-67页 |
| ·Digestion of plasmid DNAs | 第66页 |
| ·Recover of DNA fragments | 第66-67页 |
| ·Ligation of interesting gene (insert) to vector DNA | 第67页 |
| ·E. coli competent cells | 第67-68页 |
| ·Preparation of growth media | 第68-69页 |
| ·Luria-Bertani (LB) liquid medium | 第68页 |
| ·Mammalian cells culture media | 第68-69页 |
| ·SDS-PAGE | 第69-73页 |
| ·General buffers, solutions, and other reagents | 第73-75页 |
| ·General buffers | 第73-74页 |
| ·Organic reagents | 第74-75页 |
| ·The Phage display library | 第75-83页 |
| ·Growth of the library | 第75-77页 |
| ·Growth of secondary library | 第77页 |
| ·Selection on immunotubes | 第77-79页 |
| ·Further rounds of selection | 第79-80页 |
| ·Screening phage particles by ELISA | 第80-82页 |
| ·Production of soluble antibody fragments | 第82-83页 |
| ·A Practical guide for using the AdEasy system | 第83-88页 |
| ·General considerations | 第83-84页 |
| ·Generation of recombinants in bacterial cells | 第84页 |
| ·Viral production in 293 or 911 Cells | 第84-86页 |
| ·Preparation of high titer viral stocks | 第86-88页 |
| CHAPTER THREE: IDENTIFICATION OF TWO NOVEL FIBRINOLYTIC ENZYMES FROM BACILLUS SUBTILIS QK02 | 第88-113页 |
| ·Brief introduction | 第88-89页 |
| ·Materials and methods | 第89-94页 |
| ·Bacteriological technique | 第89页 |
| ·Chromatography | 第89-90页 |
| ·Enzyme assay | 第90-91页 |
| ·HPLC and SDS-PAGE | 第91页 |
| ·pH and temperature conditions | 第91页 |
| ·Inhibitors | 第91-92页 |
| ·Degradation products of fibrin | 第92页 |
| ·N-terminal amino acid sequence | 第92页 |
| ·PCR, cloning and sequencing | 第92-93页 |
| ·The protein structure homology modeling | 第93页 |
| ·Constructs and the gene expression | 第93-94页 |
| ·Results | 第94-110页 |
| ·Isolation and classification of the bacteria with the fibrinolytic activity | 第94-95页 |
| ·Purification of two fibrinolytic enzymes from B. subtilis QK02 | 第95-97页 |
| ·Fibrinolytic activity of enzymes in vitro | 第97-100页 |
| ·Enzymatic characterization | 第100-102页 |
| ·Cloning and sequences of gene encoded QK-2 | 第102-105页 |
| ·The putative three-dimensional (3D) model for subtilisin QK | 第105-109页 |
| ·Expression of qk gene encoded subtilisin QK | 第109-110页 |
| ·Discussion | 第110-113页 |
| CHAPTER FOUR: SUBTILISIN QK INHIBITS THE EXOGENOUS NITRITE AND HYDROGEN PEROXIDE INDUCED PROTEIN NITRATION, IN VITRO AND IN VIVO | 第113-129页 |
| ·Brief introduction | 第113-114页 |
| ·Meterial and methods | 第114-119页 |
| ·Materials | 第114-115页 |
| ·Detection of nitrotyrosine formation in BSA | 第115页 |
| ·Detection of structural changes of BSA in Hb/NO_2~-/H_2O_2 system | 第115-116页 |
| ·Determination of met-hemoglobin formation | 第116-117页 |
| ·Comparison of subtilisin QK with other proteins in the ability of inhibiting met-hemoglobin formation | 第117页 |
| ·Detection of protein nitration in some tissues | 第117-118页 |
| ·Cellular assay | 第118-119页 |
| ·Statistical analysis | 第119页 |
| ·Results | 第119-126页 |
| ·Subtilisin QK inhibits tyrosine nitration in BSA | 第119-120页 |
| ·Fluorescence emission spectra of tyrosine and tryptophan residues in BSA | 第120-121页 |
| ·Subtilisin QK prevent the met-hemoglobin formation | 第121-123页 |
| ·Effect of subtilisin QK on the protein nitration in some tissues of mouse, in vivo | 第123-124页 |
| ·The effect of subtilisin QK on the human umbilical vein endothelial cell injury by NaNO_2 and H_2O_2 | 第124-126页 |
| ·Discussion | 第126-129页 |
| CHAPTER FIVE: THE PLASMINOGEN ACTIVATION FUNCTION OF DegS LACKING ITS N-TERMINAL TRNSMEMBRANE DOMAIN; A NOVEL FUNCTION OF E. coli ESSENTIAL degS GENE* | 第129-143页 |
| ·Brief introduction | 第129-131页 |
| ·Meterial and methods | 第131-133页 |
| ·Strains, plasmids and cloning | 第131-132页 |
| ·Purification of recombinant protein and enzyme assay | 第132-133页 |
| ·Statistical analysis | 第133页 |
| ·Results | 第133-140页 |
| ·Amplification and sequencing of degS gene | 第133-137页 |
| ·The recombinant protein, ΔTM-DegS, acts as plasminogen activator | 第137-140页 |
| ·Discussion | 第140-143页 |
| CHAPTER SIX: IDENTIFICATION OF ANTI-FIBRIN MONOCLONIC ANTIBODY AND CONSTRUCTION OF RECOMNANT ADENOVIRUSES WITH qk AND ΔdegS GENE | 第143-162页 |
| ·Brief introduction | 第143-144页 |
| ·Meterial and methods | 第144-150页 |
| ·Library, bacteria strains, and other reagents | 第144-145页 |
| ·Preparation of human fibrin clots | 第145页 |
| ·Panning assay | 第145-146页 |
| ·Preparation of scFv-phage for monoclonal ELISA | 第146页 |
| ·Monoclonal scFv-phage ELISA | 第146-147页 |
| ·DNA sequencing assay | 第147页 |
| ·ScFv expression and Western-blot assay | 第147-148页 |
| ·ScFv production and purification | 第148页 |
| ·Purified scFv ELISA | 第148-149页 |
| ·Preparation of Competent Cells and Plasmid DNAs | 第149页 |
| ·Generation of Recombinant Adenoviral Plasmids by Homologous Recombination in E. coli | 第149-150页 |
| ·Results | 第150-159页 |
| ·Selection of fibrin specific binding clones from phage display library | 第150-151页 |
| ·Analysis of DNA sequences of the positive clones | 第151-153页 |
| ·Analysis the expression of soluble ScFv by SDS-PAGE and Western blot | 第153页 |
| ·Purification of soluble scFv by His-bond Ni affinity chromatography | 第153-154页 |
| ·Analysis of the binding activity of Purified scFv to fibrinogen and fibrin | 第154页 |
| ·Construction of eukaryotic recombinant vector pIRES-egfp-qk and pIRES-egfp-deg | 第154-156页 |
| ·Construction of the recombinant shuttle vectors for qk and degS genes | 第156-158页 |
| ·Homologous recombination in E. coli BJ5183 cells | 第158-159页 |
| ·Discussion | 第159-162页 |
| CONCLUSIONS (中文) | 第162-164页 |
| FINDING AND CREATIVITY (中文) | 第164-165页 |
| REFERENCES | 第165-189页 |
| PUBLICATIONS WITHIN STUDING AS PHD CANDIDATE | 第189-190页 |
| ACKNOWLEDGEMENT (中文) | 第190-192页 |
