| Abstract | 第3-6页 |
| 摘要 | 第7-16页 |
| List of abbreviations | 第16-18页 |
| Chapter 1. Review of literature | 第18-83页 |
| 1. Embryo development | 第18-19页 |
| 2. Stem Cell Sources and Pluripotency | 第19-21页 |
| 2.1 Embryonic Stem Cells | 第19-20页 |
| 2.2 Adult Stem Cells | 第20-21页 |
| 2.3 Induced Pluripotent Stem Cells | 第21页 |
| 2.4 Trans-differentiation | 第21页 |
| 3. Pluirpotency mechanism and its controlling network | 第21-25页 |
| 4. Importance and limitations of Pluripotent Cells | 第25-26页 |
| 5. Cellular Reprogramming | 第26-35页 |
| 5.1 Methods of Reprogramming | 第27-35页 |
| 5.1.1 Somatic Cell Nuclear Transfer(SCNT) | 第28-30页 |
| 5.1.2 Cell Fusion | 第30-32页 |
| 5.1.3 Whole Cell Extracts | 第32-33页 |
| 5.1.4 Controlling the expression of the master transcription factors | 第33-35页 |
| 6. Induced pluripotency | 第35-49页 |
| 6.1 Variable parameters of the induced pluripotency | 第37-48页 |
| 6.1.1 Choice of Reprogramming Factors | 第37-38页 |
| 6.1.2 Choice of Cell Type | 第38-39页 |
| 6.1.3 Factors delivery into target cells | 第39-45页 |
| 6.1.3.1 Viral transduction of the transgenes of defined transcription Factors | 第39-41页 |
| 6.1.3.2 Non-Viral introduction of transgenes encoding the reprogramming factors | 第41-42页 |
| 6.1.3.3 Transgene-Free Reprogramming | 第42-45页 |
| 6.1.3.3.1. Protein-Based Reprogramming | 第42-44页 |
| 6.1.3.3.2. Reprogramming Mediated by mRNA Encoding Defined Factors | 第44-45页 |
| 6.1.3.3.3. Pluripotency induction by microRNAs | 第45页 |
| 6.1.4 Culture and derivation conditions | 第45-48页 |
| 6.1.4.1 Culture conditions during reprogramming | 第45-46页 |
| 6.1.4.2 Small Molecules | 第46-48页 |
| 6.2 Identification and characterization of the reprogrammed pluripotent Cells | 第48-49页 |
| 7. Molecular mechanism of induced pluripotency | 第49-53页 |
| 8. Epigenetics and reprogramming | 第53-62页 |
| 8.1 Epigenetic code | 第53-54页 |
| 8.2 Epigenetic modifications during reprogramming to pluripotency | 第54-62页 |
| 8.2.1 Global chromatin reorganization in pluripotency reprogramming | 第57页 |
| 8.2.2 Roles of DNA modifications in ESC maintenance and reprogramming to pluripotency | 第57-61页 |
| 8.2.3 Histone modification and remodeling during iPS reprogramming | 第61-62页 |
| 9. New scientific streams have emerged from reprogramming technology | 第62-67页 |
| 9.1 Cell Replacement for Regenerative Medicine | 第63-64页 |
| 9.2 Tissue Engineering | 第64页 |
| 9.3 Disease modeling and disease physiopathology | 第64-65页 |
| 9.4 Drug Discovery and Toxicity Assessment | 第65-66页 |
| 9.5 Limitations and Perspective | 第66-67页 |
| References | 第67-83页 |
| Chapter 2. Molecular cloning of the pluripotency genes. | 第83-100页 |
| 1. Introduction | 第83-85页 |
| 2. Material and methods | 第85-94页 |
| 2.1 RNA extraction | 第85-87页 |
| 2.2 cDNA synthesis | 第87页 |
| 2.3 cDNA quality assessment | 第87-88页 |
| 2.4 Primer design and PCR amplification | 第88-90页 |
| 2.5 Gel purification of PCR Products | 第90页 |
| 2.6 Ligation of Genes into cloning vector | 第90-91页 |
| 2.7 Preparation of the competent cells for transformation | 第91页 |
| 2.8 Transformation of Ligated DNA into Bacterial Cells | 第91-92页 |
| 2.9 Colonies pick up,screening for positive colonies and plasmid extraction | 第92-93页 |
| 2.10 Screening for Positive Colonies | 第93页 |
| 2.11 DNA Sequencing to Confirm Correct DNA Identity | 第93-94页 |
| 3. Results | 第94-97页 |
| 3.1 PCR amplification of the interested factors | 第94-95页 |
| 3.2 Cloning of factors into the simple T vector | 第95-96页 |
| 3.3 Confirmation of the successful cloning of the desired genes | 第96-97页 |
| 4. Discussion | 第97-98页 |
| References | 第98-100页 |
| Chapter 3. In vitro transcription and assessment of muline pluripotency transcrlption factors'mRNAs | 第100-114页 |
| 1. Introduction | 第100-102页 |
| 2. Mate rials and methods | 第102-105页 |
| 2.1 Restriction Enzyme Digestion & Purification of the interested products | 第102页 |
| 2.2 Ligation of Gene into Expression Vector | 第102页 |
| 2.3 Bacterial transformation,colonies pick up and screening of the positive colonies | 第102-103页 |
| 2.4 In vitro transcription of mRNAs of the four transcription factors(Oct4,Sox2,c-Myc and Klf4) | 第103-104页 |
| 2.4.1 Linearization of the recombinant plasmids | 第103-104页 |
| 2.4.2 Capped transcription reaction assembly | 第104页 |
| 2.4.3 Poly(A)tailing procedure | 第104页 |
| 2.5 Recovery of the synthesized RNA | 第104-105页 |
| 2.6 In vitro assessment of the synthesized mRNAs | 第105页 |
| 3 Results | 第105-110页 |
| 3.1 Construction of the expression plasmids containing the transcription factors | 第105-107页 |
| 3.2 In vitro transcription of the desired factors | 第107-108页 |
| 3.3 Evaluation of the synthesized mRNAs | 第108-110页 |
| 4. Discussion | 第110-111页 |
| References | 第111-114页 |
| Chapter 4. Reprogramming of mouse embryonic fibroblast cells by mRNAs transfection | 第114-140页 |
| 1. Introduction | 第114-116页 |
| 2. Materials and methods | 第116-124页 |
| 2.1 Mouse Embryonic fibroblast(MEF)Isolation | 第116页 |
| 2.2 Cell Culture | 第116-117页 |
| 2.3 Cell Harvesting | 第117-118页 |
| 2.4 Cell freezing | 第118页 |
| 2.5 Cell Thawing and Recovery | 第118-119页 |
| 2.6 Cell transfection | 第119页 |
| 2.7 Flourescence Activated Cell Sorting(FACS) | 第119页 |
| 2.8 Alkaline phosphatase staining | 第119-120页 |
| 2.9 Immuno-fluorescence | 第120页 |
| 2.10 Quantitative real-Time PCR(qPCR) | 第120-121页 |
| 2.11 Bisulfite Genomic Sequencing | 第121-122页 |
| 2.12 In Vitro Differentiation of mRNA iPSCs | 第122页 |
| 2.13 Teratoma Formation and Histological Analysis | 第122-123页 |
| 2.14 Apoptosis and dead cell detection | 第123-124页 |
| 2.15 Karyotyping of the cells | 第124页 |
| 3. Results | 第124-135页 |
| 3.1 Isolation of mouse embryonic fibroblast cells | 第124-125页 |
| 3.2 Optimization of the transfection conditions | 第125-126页 |
| 3.3 Kinetics and stability monitoring of the intracellular expressed proteins | 第126-127页 |
| 3.4 Subcellular localization of the transfected factors | 第127-130页 |
| 3.5 Morphological changes during reprogramming process | 第130页 |
| 3.6 Morphological characterization of the generated colonies | 第130-131页 |
| 3.7 Immunofluorescence identification of the generated colonies | 第131-132页 |
| 3.8 Molecular characterization of the generated pluripotent like cells | 第132-133页 |
| 3.9 The developmental potential of the derived pluripotent like cells | 第133-135页 |
| 4. Discussion | 第135-136页 |
| References | 第136-140页 |
| Chapter 5. Genetic and epigenetic changes during reprogramming by mRNAs | 第140-163页 |
| 1. Introduction | 第140-141页 |
| 2. Materials and methods | 第141-145页 |
| 2.1 Cell culture | 第141页 |
| 2.2 Cell transfection | 第141-142页 |
| 2.3 Immuno-fluorescence | 第142页 |
| 2.4 Quantitative real-Time PCR(qPCR) | 第142页 |
| 2.5 Genomic DNA extraction | 第142页 |
| 2.6 Bisulfite conversion(C-T conversion) of DNA | 第142-143页 |
| 2.7 PCR amplification of the desired product(promoter region) | 第143-144页 |
| 2.8 Gel purification of PCR products and its cloning | 第144页 |
| 2.9 Methylation status analysis | 第144-145页 |
| 3. Results | 第145-157页 |
| 3.1 Expression level changes in the introduced factors and pluripotency markers during reprogramming | 第145页 |
| 3.2 Mesenchymal-to-epithelial transition(MET)during reprogramming | 第145-148页 |
| 3.3 Changes in methylation status of pluripotency factors' promoters during reprogramming | 第148-157页 |
| 4. Discussion | 第157-160页 |
| References | 第160-163页 |
| Chapter 6. Effect of some small molecules on the genetic changes duringreprogramming | 第163-175页 |
| 1. Introduction | 第163-165页 |
| 2. Materials and methods | 第165页 |
| 2.1 Cell culture | 第165页 |
| 2.2 Cell transfection | 第165页 |
| 2.3 Quantitative real-Time PCR(qPCR) | 第165页 |
| 2.4 Alkaline phosphatase staining | 第165页 |
| 3. Results | 第165-170页 |
| 4. Discussion | 第170-172页 |
| References | 第172-175页 |
| Conclusion | 第175-176页 |
| Acknowledgements | 第176-177页 |
| Publications | 第177-178页 |
