| 摘要 | 第5-7页 |
| abstract | 第7-9页 |
| Abbreviations | 第21-24页 |
| CHAPTER ⅠGENERAL INTRODUCTION | 第24-47页 |
| 1.1 IMPORTANT OF FOLLISTATIN | 第24页 |
| 1.2 MUSCLE GROWTH FACTORS | 第24-25页 |
| 1.3 MUSCLE REGULATORY FACTORS | 第25-27页 |
| 1.3.1 MYOD1/MYF-3 | 第25-26页 |
| 1.3.2 MYF-5 | 第26页 |
| 1.3.3 MYOGENIN | 第26-27页 |
| 1.3.4 MRF4 | 第27页 |
| 1.4 TRANSFORMING GROWTH FACTOR-B FAMILY | 第27-32页 |
| 1.4.1 STRUCTURE OF TGF-B FAMILY MEMBERS | 第27-28页 |
| 1.4.2 ACTIVINS AND INHIBINS STRUCTURE | 第28-30页 |
| 1.4.2.1 ACTIVIN A BIOLOGY | 第29-30页 |
| 1.4.3 OTHER MEMBERS OF TGF-B FAMILY | 第30-32页 |
| 1.4.3.1 MYOSTATIN IS A POTENTIAL BINDING PARTNER OF FOLLISTATIN | 第30页 |
| 1.4.3.2 STRUCTURE OF MYOSTATIN | 第30-31页 |
| 1.4.3.2 MYOSTATIN NEGATIVELY REGULATES MUSCLE GROWTH | 第31-32页 |
| 1.5 FOLLSTATIN STRUCTURAL CHARACTERIZATION | 第32-36页 |
| 1.5.1 THE FOLLISTATIN GENE | 第32页 |
| 1.5.2 FOLLISTATIN ISOFORMS FROM ALTERNATIVE PRE-MRNA SPLICING | 第32-33页 |
| 1.5.3 FOLLISTATIN DOMAINS | 第33-34页 |
| 1.5.4 ROLE OF FOLLISTATIN AS A BINDING PROTEIN | 第34-35页 |
| 1.5.4.1 FOLLISTATIN IS A ACTIVIN BINDING PROTEIN | 第34页 |
| 1.5.4.2 AFFINITY TO HEPARAN SULFATE PROTEOGLYCANS | 第34页 |
| 1.5.4.3 FOLLISTATIN BIND OTHER TGF-B MEMBERS PROTEIN | 第34-35页 |
| 1.5.5 DIFFERENCES BETWEEN FOLLISTATIN 288 AND 315 | 第35-36页 |
| 1.5.5.1 DIFFERENCE BETWEEN FOLLISTATIN ISOFORMS DISTRIBUTION IN EXPRESSION | 第35页 |
| 1.5.5.2 DIFFERENCE AFFINITY OF FOLLISTATIN ISOFORMS FOR ACTIVINS | 第35-36页 |
| 1.6 ROLE OF FOLLISTATIN IN MUSCLE GROWTH | 第36页 |
| 1.7 FOLLISTATIN OVER-EXPRESSION | 第36-41页 |
| 1.7.1 EFFECTS OF FOLLISTATIN OVER-EXPRESSION ON MUSCLE | 第36-38页 |
| 1.7.2 EFFECTS OF OVER-EXPRESSION OF FOLLISTATIN ON OVARIAN FUNCTION | 第38页 |
| 1.7.3 EFFECTS OF OVER-EXPRESSION OF FOLLISTATIN ON TESTIS FUNCTION | 第38-39页 |
| 1.7.4 FOLLISTATIN OVER-EXPRESSION BY ADENO-ASSOCIATED VIRUS | 第39-41页 |
| 1.7.4.1 ADVANTAGES OF RECOMBINANT ADENO-ASSOCIATED VIRUS FOR GENE DELIVERYAND EXPRESSION | 第39-40页 |
| 1.7.4.2 FOLLISTATIN OVER-EXPRESSION BY AAV ENHANCES MUSCLE GROWTH ANDSTRENGTH | 第40-41页 |
| 1.8 ADENO-ASSOCIATED VIRUSES(AAVS)STRUCTURE | 第41-43页 |
| 1.8.1 AAV GENOME,TRANSCRIPTOME AND PROTEOME | 第41页 |
| 1.8.2 ITR SEQUENCES | 第41-42页 |
| 1.8.3 REP GENES AND REP PROTEINS | 第42页 |
| 1.8.4 CAP GENES AND VP PROTEINS | 第42-43页 |
| 1.9 AAV SEROTYPES,RECEPTORS AND NATIVE TROPISM | 第43-44页 |
| 1.9.1 SEROTYPE 2 | 第43页 |
| 1.9.2 OTHER SEROTYPES | 第43-44页 |
| 1.10 CONCLUSION | 第44-46页 |
| 1.11 OBJECTIVES | 第46-47页 |
| CHAPTER Ⅱ OVINE PRIMARY MYOBLAST CELL CULTURE AND ISOLATION | 第47-59页 |
| 2.1 INTRODUCTION | 第47页 |
| 2.2 MATERIALS AND METHODS | 第47-53页 |
| 2.2.1 CHEMICAL AND MEDIUM | 第47-48页 |
| 2.2.2 PRIMARY MYOBLAST CULTURES | 第48-49页 |
| 2.2.3 THAWING CELLS | 第49-50页 |
| 2.2.4 DETERMINING CELL DENSITY AND VIABILITY | 第50页 |
| 2.2.5 FREEZING CELLS | 第50页 |
| 2.2.6. PRIMARY MYOBLAST CELLS GROWTH CURVE | 第50页 |
| 2.2.7 CELL STAINING | 第50-51页 |
| 2.2.8 RNA ISOLATION | 第51页 |
| 2.2.9 RNA GEL QUANTIFICATION | 第51-52页 |
| 2.2.10 COMPLEMENTARY DNA SYNTHESIS | 第52页 |
| 2.2.11 RT-PCR | 第52-53页 |
| 2.2.12 IMMUNOFLUORESCENCE STAINING | 第53页 |
| 2.3 RESULTS AND DISCUSSION | 第53-58页 |
| 2.3.1 OVINE PRIMARY MYOBLAST PRODUCTION IN VITRO | 第53-55页 |
| 2.3.2 GROWTH CURVE OF PRIMARY MYOBLAST CELLS | 第55页 |
| 2.3.3 IMMUNOFLUORESCENCE STAINING AND RT-PCR BASED IDENTIFICATION OF OPMS | 第55-57页 |
| 2.3.4 MYOGENIC INDUCTION OF OPMS | 第57-58页 |
| 2.4 CONCLUSION | 第58-59页 |
| CHAPTER Ⅲ CLONING AND SEQUENCING OF FOLLISTATIN CDNA FROM OVINE MYOBLAST ANDCONSTRUCTION OF AAV VECTOR ENCODING FOLLISTATIN | 第59-72页 |
| 3.1 INTRODUCTION | 第59页 |
| 3.2 MATERIALS AND METHODS | 第59-66页 |
| 3.2.1 NUCLEIC ACID MANIPULATION | 第59-60页 |
| 3.2.2 FOLLISTATIN GENE IDENTIFICATION | 第60-62页 |
| 3.2.2.1 RNA ISOLATION AND CDNA SYNTHESIS | 第60页 |
| 3.2.2.2 RT-PCR | 第60页 |
| 3.2.2.3 LIGATION | 第60页 |
| 3.2.2.4 PLASMID TRANSFORMATION INTO DH5ALPHA E.COLI CELLS USING HEAT SHOCK | 第60-61页 |
| 3.2.2.5 GROWTH OF TRANSFORMED E. COLI BACTERIA CULTURES | 第61页 |
| 3.2.2.6 PLASMID PURIFICATION | 第61-62页 |
| 3.2.3 CONSTRUCTION OF THE PAAV-CFS-FLAG PLASMID VECTOR | 第62页 |
| 3.2.3.1 RT-PCR | 第62页 |
| 3.2.3.2 CLONING OF OVINE FOLLISTATIN ORF INTO THE PAAV-IRIS-GFP PLASMID VECTOR | 第62页 |
| 3.2.4 PACKAGING AAV VECTORS | 第62-63页 |
| 3.2.5 TRANSDUCTION OF OVINE PRIMARY MYOBLAST CELLS | 第63页 |
| 3.2.6 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) | 第63-64页 |
| 3.2.6.1 PROTEIN GEL STAINING | 第64页 |
| 3.2.7 WESTERN BLOT ANALYSIS | 第64-65页 |
| 3.2.8 FACS ANALYSIS OF GFP EXPRESSION | 第65页 |
| 3.2.9 STATISTICAL ANALYSIS | 第65-66页 |
| 3.3 RESULTS AND DISCUSSION | 第66-70页 |
| 3.3.1 EXPRESSION ANALYSIS OF OVINE FOLLISTATIN | 第66页 |
| 3.3.2 SCREENING TRANSFORMANTS FOR INSERTS | 第66页 |
| 3.3.3 CLONING OF THE FOLLISTATIN GENE AND NUCLEOTIDE SEQUENCE ANALYSIS | 第66-67页 |
| 3.3.4 FOLLISTATIN PROTEIN IS SYNTHESIZED IN TRANSDUCED MYOBLAST CELLS | 第67页 |
| 3.3.5 TRANSDUCED MYOBLAST EXPRESS GFP PROTEIN | 第67-70页 |
| 3.3.6 VIRADUCTIN INCREASE TRANSDUCTION EFFICIENCY | 第70页 |
| 3.4 CONCLUSION | 第70-72页 |
| CHAPTER Ⅳ ADENO-ASSOCIATED VIRUS MEDIATED FOLLISTATIN OVER-EXPRESSION INDUCEMYOBLAST PROLIFERATION | 第72-92页 |
| 4.1 INTRODUCTION | 第72-73页 |
| 4.2 MATERIALS AND METHODS | 第73-77页 |
| 4.2.1 TRANSDUCTION OF PRIMARY MYOBLAST CELLS | 第73页 |
| 4.2.2 RNA ISOLATION AND RT-PCR | 第73页 |
| 4.2.3 QUANTIFICATION OF GENE EXPRESSION WITH REAL-TIME PCR | 第73-74页 |
| 4.2.3.1 INTRODUCTION TO THE IQ5 REAL-TIME PCR DETECTION SYSTEMS | 第73-74页 |
| 4.2.3.2 REAL-TIME QUANTITATIVE PCR ANALYSIS | 第74页 |
| 4.2.4 WESTERN BLOT ANALYSIS | 第74-76页 |
| 4.2.5 PROLIFERATION ASSAY | 第76页 |
| 4.2.6 CELL CYCLE ANALYSIS | 第76-77页 |
| 4.2.6.1 INTRODUCTION TO CELL CYCLE PHASES | 第76页 |
| 4.2.6.2 CELL CYCLE ANALYSIS BY DNA CONTENT(PROPIDIUM IODIDE) | 第76-77页 |
| 4.2.7 STATISTICAL ANALYSIS | 第77页 |
| 4.3 RESULTS | 第77-88页 |
| 4.3.1 EXPRESSION ANALYSIS OF OVINE FOLLISTATIN AND MYOSTATIN | 第77页 |
| 4.3.2 CHANGES IN PROTEIN CONCENTRATION DURING MYOBLAST PROLIFERATION | 第77-79页 |
| 4.3.3 CHANGES IN MRNA CONCENTRATION DURING MYOBLAST PROLIFERATION | 第79-83页 |
| 4.3.5 EFFECTS OF OVINE FST OVER-EXPRESSION ON GENE EXPRESSION | 第83页 |
| 4.3.6 FOLLISTATIN OVER-EXPRESSION INCREASES PROLIFERATION OF OVINE PRIMARYMYOBLASTS | 第83-85页 |
| 4.3.7 FST OVEREXPRESSION INDUCES PHOSPHORELATEION OF EXTRACELLULAR SIGNAL-REGULATED KINASE(ERK)1/2 AND AKT | 第85-87页 |
| 4.3.8 FST PROTEIN INTERFERES AT THE G1 PHASE OF THE CELL CYCLE | 第87-88页 |
| 4.4 DISCUSSION | 第88-91页 |
| 4.5 CONCLUSION | 第91-92页 |
| CHAPTER Ⅴ INDUCTION OF DIFFERENTIATION USING ADENO-ASSOCIATED VIRUS MEDIATEDFOLISTATIN OVER-EXPRESSION IN MYOBLASTS | 第92-102页 |
| 5.1 INTRODUCTION | 第92-93页 |
| 5.2 MATERIALS AND METHODS | 第93-94页 |
| 5.2.1 OVINE PRIMARY MYOBLAST CULTURE AND DIFFERENTIATION | 第93页 |
| 5.2.2 CELL STAINING AND DETERMINATION OF FUSION PERCENTAGE | 第93-94页 |
| 5.2.3 STATISTICAL ANALYSIS | 第94页 |
| 5.3 RESULTS | 第94-97页 |
| 5.3.1 CHANGES IN MRNA CONCENTRATION DURING MYOBLAST DIFFERENTIATION | 第94-96页 |
| 5.3.2 EFFECTS OF FOLLISTATIN OVER-EXPRESSION ON GENES EXPRESSION | 第96页 |
| 5.3.3 FOLLISTATIN OVER-EXPRESSION PROMOTES DIFFERENTIATION IN MYOBLASTS | 第96-97页 |
| 5.4 DISCUSSION | 第97-101页 |
| 5.5 CONCLUSION | 第101-102页 |
| CHAPTER Ⅵ AAV-2 VECTOR INTEGRATE INTO THE OVINE MYOBLAST GENOME RANDOMLY ANDPROMOTE DIFFERENTIATION AND PROLIFERATION | 第102-115页 |
| 6.1 INTRODUCTION | 第102-103页 |
| 6.2 MATERIALS AND METHODS | 第103-108页 |
| 6.2.1 GENOMIC DNA EXTRACTION | 第103页 |
| 6.2.2 SOUTHERN BLOT ANALYSIS | 第103-106页 |
| 6.2.3 CELL STAINING AND DETERMINATION OF FUSION PERCENTAGE | 第106页 |
| 6.2.4 PRODUCING POSITIVE TRANSGENIC MYOBLAST CELLS | 第106-107页 |
| 6.2.5 WESTERN BLOT | 第107页 |
| 6.2.6 CELL PROLIFERATION ASSAY | 第107页 |
| 6.2.7 TRANSGENIC MYOBLAST CELL CYCLE ANALYSIS | 第107页 |
| 6.2.8 STATISTICAL ANALYSIS | 第107-108页 |
| 6.3 RESULTS | 第108-113页 |
| 6.3.1 SCREENING AND ANALYSIS OF TRANSGENIC MYOBLAST CELLS | 第108-109页 |
| 6.3.2 AAV VECTOR INTEGRATES INTO THE MYOBLAST GENOME | 第109-110页 |
| 6.3.3 MULTIPLE COPY INTEGRATION INCREASES FOLLISTATIN PROTEIN EXPRESSION INTRANSGENIC CLONES | 第110页 |
| 6.3.4 PROLIFERATION AND DIFFERENTIATION ARE INDUCED IN TRANSGENIC MYOBLASTCELLS | 第110-112页 |
| 6.3.5 FST OVER-EXPRESSION INTERFERES AT THE G1 PHASE IN TRANSGENIC CELLS | 第112-113页 |
| 6.4 DISCUSSION | 第113-114页 |
| 6.5 CONCLUSION | 第114-115页 |
| CHAPTER Ⅶ SUMMARY,CONCLUSION AND SUGGESTIONS | 第115-119页 |
| 7.0 SUMMARY | 第115-116页 |
| 7.1 CONCLUSION | 第116-118页 |
| 7.2 SUGGESTIONS | 第118-119页 |
| REFERENCES | 第119-128页 |
| ACKNOWLEDGEMENTS | 第128-131页 |
| AUTHOR'S BIOGRAPHY | 第131-132页 |
