Conversion of Mouse Embryonic Fibroblast and Bone Marrow Mes
发布时间：2021-01-08 19:34
Bone is a well specialized supporting framework of the body,characterized by its rigidity,hardness,regeneration power and repair.It shields the vital organs of the body,serves as an environment for marrow,and acts as a mineral stock for calcium homeostasis and a reservoir of growth factors and cytokines.It also contributes in acid-base balance.Understanding the role of bone cells is important,not only in the orthopedic field,but also in research field involving bone.Bone cells work in harmony to...
【文章来源】：扬州大学江苏省
【文章页数】：156 页
【学位级别】：博士
【文章目录】：
Abstract
List of abbreviations
Chapter 1. Review of literature
1. Bone biology
1.1 Bone function
1.2 Bone anatomy
1.3 Bone components
1.3.1 Bone matrix （ECM）
1.3.2 Cellular components
1.3.3 Bone marrow
1.4 Bone development （formation）
1.4.1 Endochondral ossification
1.4.2 Intramembranous ossification
1.5 Bone remodeling
1.6 Bone healing
1.6.1 The inflammatory phase
1.6.2 The reparative phase
1.6.3 The remodeling phase
2. The Need for Bone Repair and Current Therapies
2.1 The need for bone repair
2.2 The current treatments for bone repair
3. Tissue engineering strategies for bone regeneration
3.1 Cell based strategies
3.1.1 Tissue specific cells （Osteoblasts）
3.1.2 Stem cells
3.1.2.1 Mesenchymal stem cells （MSCs）
3.1.2.2 Embryonic stem cells （ESCs）
3.1.2.3 Induced pluripotent stem cells （iPSCs）
3.2 Osteoinductive factors
3.2.1 Osteoinductive chemical factors
3.2.2 Osteoinductive biological factors
3.3 Biomaterials （Scaffold materials）
4. Gene therapy strategies for bone regeneration
4.1 Methods of Gene delivery
4.1.1 The in vivo gene delivery
4.1.2 The ex vivo gene delivery
4.1.2.1 Cell-mediated gene therapy
4.2 Vectors for gene delivery
4.2.1 Gene delivery via non-viral vectors
4.2.2 Gene delivery via viral vectors
4.2.2.1 Retrovirus/Lentivirus
4.2.2.2 Adenovirus
4.2.2.3 AAV
5. Lineage reprogramming
5.1 Trans-differentiation （trans-determination or direct conversion）
5.2 Direct reprogramming
6. References
Chapter 2. Conversion of mouse embryonic fibroblasts （MEF） into functional osteoblasts by defined factor
1. Introduction
2. Material and methods
2.1 Genetic materials
2.1.1 Primer design and PCR amplification
2.1.2 Gel purification of PCR Products
2.1.3 Ligation of Genes into cloning vector
2.1.4 Restriction Enzyme Digestion & Purification of the interested products
2.1.5 Preparation of the competent cells for transformation
2.1.6 Transformation of Ligated DNA into Bacterial Cells
2.1.7 Colonies pick up, screening for positive colonies and plasmid extraction
2.1.8 Screening for Positive Colonies
2.1.9 DNA Sequencing to Confirm Correct DNA Identity
2.1.10 Construction of the Lentivirus expression vectors
2.2 Cells
2.2.1 Isolation of MEF
2.2.2 Cell Culture
2.2.3 Cell Harvesting
2.2.4 Cell freezing
2.2.5 Cell Thawing and Recovery
2.3 Study design
2.4 In vitro osteogenic Induction
2.4.1 Determination of the multiplicity of infection （MOI）
2.4.2 Lentivirus mediated transduction
2.5 Characterization of the osteogenic induction
2.5.1 Real-Time RT-PCR
2.5.1.1 RNA extraction
2.5.1.2 cDNA synthesis
2.5.1.3 RTPCR reaction
2.5.2 In vitro mineralization assay
2.5.2.1 ALP staining
2.5.2.2 Alizarin Red staining
2.5.2.3 Von Kossa staining
2.5.3 Fluorescence Immunocytochemistry
2.5.4 Western blot analysis
2.6 Statistical analysis
3. Results
3.1 PCR amplification of the interested factors
3.2 Confirmation of the successful cloning of the desired genes
3.3 Confirmation of the correct DNA Identity
3.4 Isolation and culture of MEF cells
3.5 Lentivirus transduction efficiency and the multiplicity of infection
3.6 Induction of osteoblasts from MEF by the combination of hLMP-3 and Yamanaka factors
3.6.1 Morphological changes during reprogramming process
3.6.2 Molecular characterization of the hLMP-3 in the transduced cells
3.6.3 Expression level changes of the bone gene markers during reprogramming
3.7 Conversion efficiency of induced osteoblasts from c-Myc, Oct4 and hLMP-3 combination
3.7.1 Characterization of the generated osteoblasts using in vitro mineralization assay
3.7.2 Evaluation of OCN expression as a late osteogenic marker
3.8 MEF cells were directly reprogrammed into osteoblast like cells without passing through anintermediate pluripotency stage
4. Discussion
5. References
Chapter 3. In vivo study of the osteoblast formed cells after direct reprogramming of MEFs to form new bone inan induced bone defect
1. Introduction
2. Material and methods
2.1 Animal model
2.2 MEF cells isolation, transduction, and characterization
2.3 Cell seeding and scaffold material
2.4 Animal anesthesia and surgical procedures:
2.5 Evaluation of the transplantation procedure
2.5.1 Radiographic examination
2.5.2 Histological examination
3. Results
3.1 Induction of a unilateral cortical femoral defect in SD rat
3.2 Osteoblasts from the COL group induced bone repair after transplantation into a unilateralbone defect
4. Discussion
5. References
Chapter 4.Comparison between curcumin and all-trans retinoic acid in the osteogenic differentiation of mouse bonemarrow mesenchymal stem cells
1. Introduction
2. Material and methods
2.1 Experimental animals
2.2 Cells
2.2.1 Isolation and culture of mouse BMSCs
2.2.2 BMSCs freezing, thawing and recovery
2.2.3 Characterization of mouse BMSCs
2.3 Study design
2.4 In vitro osteogenic Induction
2.4.1 Experiment （Ⅰ）
2.4.2 Experiment （Ⅱ）
2.5 Characterization of the osteogenic differentiation
2.5.1 Real-Time RT PCR
2.5.2 In vitro mineralization assay
2.5.3 Fluorescence Immunocytochemistry
2.5.4 Western blot analysis
2.6 Statistical analysis
3. Results
3.1 Morphological and immunotyping characterization of mouse BMSCs
3.2 Morphological changes during the osteogenic differentiation of BMSCs
3.3 Osteogenic differentiation capacity of BMSCs after induction with curcumin and ATRA
3.4 The effect of curcumin and ATRA on the expression level of the bone associated gene markersduring BMSCs osteogenic differentiation
3.5 Expression of the OCN during the osteogenic differentiation process
3.6 Effect of curcumin supplemented OM on the osteogenic differentiation of lentivirustransduced MEFs with hLMP-3
4. Discussion
5. References
Conclusion
Innovations
Acknowledgements
Publications
【参考文献】：
期刊论文
[1]Mesenchymal stem cells: Molecular characteristics and clinical applications[J]. Farbod Rastegar,Deana Shenaq,Eric R Wagner,Stephanie H Kim,Russell R Reid,Hue H Luu,Rex C Haydon. World Journal of Stem Cells. 2010(04)
本文编号：2965173
【文章来源】：扬州大学江苏省
【文章页数】：156 页
【学位级别】：博士
【文章目录】：
Abstract
List of abbreviations
Chapter 1. Review of literature
1. Bone biology
1.1 Bone function
1.2 Bone anatomy
1.3 Bone components
1.3.1 Bone matrix （ECM）
1.3.2 Cellular components
1.3.3 Bone marrow
1.4 Bone development （formation）
1.4.1 Endochondral ossification
1.4.2 Intramembranous ossification
1.5 Bone remodeling
1.6 Bone healing
1.6.1 The inflammatory phase
1.6.2 The reparative phase
1.6.3 The remodeling phase
2. The Need for Bone Repair and Current Therapies
2.1 The need for bone repair
2.2 The current treatments for bone repair
3. Tissue engineering strategies for bone regeneration
3.1 Cell based strategies
3.1.1 Tissue specific cells （Osteoblasts）
3.1.2 Stem cells
3.1.2.1 Mesenchymal stem cells （MSCs）
3.1.2.2 Embryonic stem cells （ESCs）
3.1.2.3 Induced pluripotent stem cells （iPSCs）
3.2 Osteoinductive factors
3.2.1 Osteoinductive chemical factors
3.2.2 Osteoinductive biological factors
3.3 Biomaterials （Scaffold materials）
4. Gene therapy strategies for bone regeneration
4.1 Methods of Gene delivery
4.1.1 The in vivo gene delivery
4.1.2 The ex vivo gene delivery
4.1.2.1 Cell-mediated gene therapy
4.2 Vectors for gene delivery
4.2.1 Gene delivery via non-viral vectors
4.2.2 Gene delivery via viral vectors
4.2.2.1 Retrovirus/Lentivirus
4.2.2.2 Adenovirus
4.2.2.3 AAV
5. Lineage reprogramming
5.1 Trans-differentiation （trans-determination or direct conversion）
5.2 Direct reprogramming
6. References
Chapter 2. Conversion of mouse embryonic fibroblasts （MEF） into functional osteoblasts by defined factor
1. Introduction
2. Material and methods
2.1 Genetic materials
2.1.1 Primer design and PCR amplification
2.1.2 Gel purification of PCR Products
2.1.3 Ligation of Genes into cloning vector
2.1.4 Restriction Enzyme Digestion & Purification of the interested products
2.1.5 Preparation of the competent cells for transformation
2.1.6 Transformation of Ligated DNA into Bacterial Cells
2.1.7 Colonies pick up, screening for positive colonies and plasmid extraction
2.1.8 Screening for Positive Colonies
2.1.9 DNA Sequencing to Confirm Correct DNA Identity
2.1.10 Construction of the Lentivirus expression vectors
2.2 Cells
2.2.1 Isolation of MEF
2.2.2 Cell Culture
2.2.3 Cell Harvesting
2.2.4 Cell freezing
2.2.5 Cell Thawing and Recovery
2.3 Study design
2.4 In vitro osteogenic Induction
2.4.1 Determination of the multiplicity of infection （MOI）
2.4.2 Lentivirus mediated transduction
2.5 Characterization of the osteogenic induction
2.5.1 Real-Time RT-PCR
2.5.1.1 RNA extraction
2.5.1.2 cDNA synthesis
2.5.1.3 RTPCR reaction
2.5.2 In vitro mineralization assay
2.5.2.1 ALP staining
2.5.2.2 Alizarin Red staining
2.5.2.3 Von Kossa staining
2.5.3 Fluorescence Immunocytochemistry
2.5.4 Western blot analysis
2.6 Statistical analysis
3. Results
3.1 PCR amplification of the interested factors
3.2 Confirmation of the successful cloning of the desired genes
3.3 Confirmation of the correct DNA Identity
3.4 Isolation and culture of MEF cells
3.5 Lentivirus transduction efficiency and the multiplicity of infection
3.6 Induction of osteoblasts from MEF by the combination of hLMP-3 and Yamanaka factors
3.6.1 Morphological changes during reprogramming process
3.6.2 Molecular characterization of the hLMP-3 in the transduced cells
3.6.3 Expression level changes of the bone gene markers during reprogramming
3.7 Conversion efficiency of induced osteoblasts from c-Myc, Oct4 and hLMP-3 combination
3.7.1 Characterization of the generated osteoblasts using in vitro mineralization assay
3.7.2 Evaluation of OCN expression as a late osteogenic marker
3.8 MEF cells were directly reprogrammed into osteoblast like cells without passing through anintermediate pluripotency stage
4. Discussion
5. References
Chapter 3. In vivo study of the osteoblast formed cells after direct reprogramming of MEFs to form new bone inan induced bone defect
1. Introduction
2. Material and methods
2.1 Animal model
2.2 MEF cells isolation, transduction, and characterization
2.3 Cell seeding and scaffold material
2.4 Animal anesthesia and surgical procedures:
2.5 Evaluation of the transplantation procedure
2.5.1 Radiographic examination
2.5.2 Histological examination
3. Results
3.1 Induction of a unilateral cortical femoral defect in SD rat
3.2 Osteoblasts from the COL group induced bone repair after transplantation into a unilateralbone defect
4. Discussion
5. References
Chapter 4.Comparison between curcumin and all-trans retinoic acid in the osteogenic differentiation of mouse bonemarrow mesenchymal stem cells
1. Introduction
2. Material and methods
2.1 Experimental animals
2.2 Cells
2.2.1 Isolation and culture of mouse BMSCs
2.2.2 BMSCs freezing, thawing and recovery
2.2.3 Characterization of mouse BMSCs
2.3 Study design
2.4 In vitro osteogenic Induction
2.4.1 Experiment （Ⅰ）
2.4.2 Experiment （Ⅱ）
2.5 Characterization of the osteogenic differentiation
2.5.1 Real-Time RT PCR
2.5.2 In vitro mineralization assay
2.5.3 Fluorescence Immunocytochemistry
2.5.4 Western blot analysis
2.6 Statistical analysis
3. Results
3.1 Morphological and immunotyping characterization of mouse BMSCs
3.2 Morphological changes during the osteogenic differentiation of BMSCs
3.3 Osteogenic differentiation capacity of BMSCs after induction with curcumin and ATRA
3.4 The effect of curcumin and ATRA on the expression level of the bone associated gene markersduring BMSCs osteogenic differentiation
3.5 Expression of the OCN during the osteogenic differentiation process
3.6 Effect of curcumin supplemented OM on the osteogenic differentiation of lentivirustransduced MEFs with hLMP-3
4. Discussion
5. References
Conclusion
Innovations
Acknowledgements
Publications
【参考文献】：
期刊论文
[1]Mesenchymal stem cells: Molecular characteristics and clinical applications[J]. Farbod Rastegar,Deana Shenaq,Eric R Wagner,Stephanie H Kim,Russell R Reid,Hue H Luu,Rex C Haydon. World Journal of Stem Cells. 2010(04)
本文编号：2965173
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