可MMP酶解并可缓释出BMP-2新蚕丝材料的创制与研究
发布时间：2021-10-25 06:46
蚕丝作为一种来源于家蚕（Bombyx mori）的天然高分子材料,以其优异的性能而闻名,并因其作为可应用于骨组织工程的生物材料而受到人们的关注。然而天然蚕丝材料尚不能满足骨组织工程生物材料某些特定的需求,例如天然丝材料缺乏骨相关生长因子与合适降解速率。骨形态发生蛋白（Bone Morphogenetic Protein,BMP-2）是骨组织再生过程中不可或缺的生长因子,其已经被广泛应用于蚕丝生物材料研究领域。迄今为止,不同的偶联方案已被用于赋予多孔3D海绵、膜、微粒和电纺丝垫等丝素创制材料新功能和能够释放出BMP-2。丝素生物材料与BMP-2的偶联多采用非共价偶联的策略,这种结合方式在生物材料使用的最初几天易引起BMP-2发生“突释”现象。因此,如何能够在赋予蚕丝生物材料新功能情况下并能够控制功能蛋白或因子的释放成为这类蚕丝材料应用研究的热点和难点。虽然通过直接添食、化学改性、中尺度组装和大尺度混合等改性方法,能够实现赋予蚕丝材料新的所需功能,但是这些策略需要每次使用时对材料进行复杂的改性实验而且很难保证获得的每批材料的改性一致性。家蚕种系转化（germline transformat...
【文章来源】：西南大学重庆市 211工程院校 教育部直属院校
【文章页数】：135 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
CHAPTER Ⅰ.LITTERATURE REVIEW
1.1 THE ATTRACTIVENESS OF SF FOR TISSUE ENGINEERING APPLICATIONS
1.2 BONE TISSUE IS ACTIVELY RESEARCHED IN THE FIELD OF TISSUE ENGINEERING
1.3 BONE DEFECTS AND THERAPY
1.4 BONE TISSUE ENGINEERING APPROACH
1.4.1 Requirements for bone tissue engineering scaffold
1.5 BONE TISSUE ENGINEERING VIA GROWTH FACTOR DELIVERY
1.5.1 Identifying the most potent bone growth factor
1.5.2 Delivery system for BMP-
1.5.3 Silk fibroin,drug delivery system for BMP-
1.5.4 Burst release phenomenon
1.5.5 Controlled release of BMP-2 from silk fibroin materials
1.5.6 Smart drug delivery:release on demand
1.6 BIODEGRADATION OF SILK FIBROIN,A PREREQUISITE FEATURE TO BE TAKEN INTO CONSIDERATION
1.6.1 Degradation behaviour of silk fibroin
1.6.2 Strategies for controlling the degradation rate of SF
1.6.3 Acute inflammation responses and macrophage-mediated degradation
1.6.4 MMP-degradable silk fibroin
CHAPTER Ⅱ.GENERAL INTRODUCTION
2.1 RESEARCH BACKGROUND
2.2 RESEARCH SIGNIFICANCE
2.2.1 Burst release effects and controlled release approach
2.2.2 Biodegradation of silk materials,an important feature to be taken into considerations
2.3 RESEARCH OBJECTIVE
2.4 RESEARCH CONTENT
2.5 WORKFLOW OF THE STUDY
CHAPTER Ⅲ.TRANSGENIC SILKWORMS PRODUCING HUMAN BONE MORPHOGENETIC GROWTH FACTOR2 FUSED TO FIBROIN HEAVY CHAIN VIA MMP SENSITIVE LINKERS
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 Materials
3.2.2 Major instruments and Appliances
3.2.3 General reagents and kits
3.2.4 Media,buffers and solutions setup
3.3 METHODOLOGICAL SECTIONS
3.3.1 Primer design and synthesis
3.3.2 Design and synthesis of hBMP-2 derived genes with MMP linkers at both termini
3.3.3 Plate streaking,bacteria growth and plasmid extraction
3.3.4 Inclusion of MMP sensitive linkers
3.3.5 Construction of the expression cassette and subsequent cloning into piggyBac vector
3.3.6 Construction of recombinant piggyBac vectors
3.3.7 Embryo preparation and microinjection
3.3.8 Embryo development and larvae rearing
3.3.9 Screening and identification of the transgenic silkworms
3.3.10 Validation of transgene integration into silkworm genome
3.3.11 Identification and quantification of recombinant protein from silkworm cocoons
3.4 RESULTS AND DISCUSSIONS
3.4.1 BMP-2 fused to fibroin heavy-chain gene via matrix metalloproteinase sensitive linkers presenting different catalytic activities
3.4.2 Transgenic silkworms expelling BMP-2 as a foreign constituent of the cocoon fiber
3.4.3 Transgenic silkworm lines producing BMP-2 tethered with Matrix metalloproteinase sensitive linkers
3.5 CONCLUSION
CHAPTER Ⅳ.IN VITRO DEGRADATION OF SILK FIBROIN BY MMP-2 ENZYMES SECRETED BY MACROPHAGES
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.2.1 Biological materials
4.2.2 Instrument and equipment
4.2.3 Chemical reagents
4.3 METHODOLOGICAL SECTION
4.3.1 Fabrication of silk based materials
4.3.2 Quantitation of the level of BMP-2 in different silk fibroin materials
4.3.3 Sterilization of silk fibroin slices
4.3.4 Culture of human THP-1 monocyte cells
4.3.5 Differentiation of monocyte THP1 and polarization into pro-inflammatory M1 macrophages
4.3.6 In vitro macrophage-mediated degradation of silk fibroin slices In vitro MMP-mediated degradation system set up
4.3.7 Statistical analysis
4.4 RESULTS
4.4.1 Regeneration processes decreased the content of BMP-2 at certain extent
4.4.2 Macrophages are the prevalent cell type observed on silk fibroin implants and can serve as the source of MMP-2 enzyme
4.4.3 The incorporation of MMP cleavable substrate presenting various kinetic activity into silk fibroin results in different degradation rate
4.4.4 The addition of MMP cleavable peptide promoted the degradation of SF slices
4.5 Conclusions
CHAPTER Ⅴ.IN VITRO MMP-MEDIATED RELEASE AND BIOACTIVITY OF RELEASED BMP-
5.1 INTRODUCTION
5.2 MATERIALS
5.2.1 Cell lines
5.2.2 Major equipment
5.2.3 Reagents and kits
5.3 METHODOLOGICAL SECTION
5.3.1 Generation of silk fibroin aqueous solution
5.3.2 Preparation of macrophage conditioned media:source of MMP-
5.3.3 Ascertain the susceptibility of peptide sites and cleavage by MMP-
5.3.4 In vitro macrophage-mediated release
5.3.5 Quantification of BMP-2 from release media
5.3.6 Quantification of BMP-2 in remained silk fibroin slices
5.3.7 Bioactivity of BMP-2 released from cocoon slices
5.3.8 Statistical analysis
5.4 RESULTS AND DISCUSSIONS
5.4.1 Digestion of cleavable linkers by MMP secreted by macrophages freed BMP-2 out from silk fibroin
5.4.2 The cleavage activity occurred at different rate resulting in tunable release profile
5.4.3 BMP-2 released from transgenic silk fibroin groups stimulated MSC proliferation
5.5 CONCLUSION
CHAPTER Ⅵ.SUMMARY AND CONCLUSIONS
论文创新点
REFERENCES
在读期间发表文章、参加会议及参研课题
ACKNOWLDEMENT
本文编号：3456862
【文章来源】：西南大学重庆市 211工程院校 教育部直属院校
【文章页数】：135 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
CHAPTER Ⅰ.LITTERATURE REVIEW
1.1 THE ATTRACTIVENESS OF SF FOR TISSUE ENGINEERING APPLICATIONS
1.2 BONE TISSUE IS ACTIVELY RESEARCHED IN THE FIELD OF TISSUE ENGINEERING
1.3 BONE DEFECTS AND THERAPY
1.4 BONE TISSUE ENGINEERING APPROACH
1.4.1 Requirements for bone tissue engineering scaffold
1.5 BONE TISSUE ENGINEERING VIA GROWTH FACTOR DELIVERY
1.5.1 Identifying the most potent bone growth factor
1.5.2 Delivery system for BMP-
1.5.3 Silk fibroin,drug delivery system for BMP-
1.5.4 Burst release phenomenon
1.5.5 Controlled release of BMP-2 from silk fibroin materials
1.5.6 Smart drug delivery:release on demand
1.6 BIODEGRADATION OF SILK FIBROIN,A PREREQUISITE FEATURE TO BE TAKEN INTO CONSIDERATION
1.6.1 Degradation behaviour of silk fibroin
1.6.2 Strategies for controlling the degradation rate of SF
1.6.3 Acute inflammation responses and macrophage-mediated degradation
1.6.4 MMP-degradable silk fibroin
CHAPTER Ⅱ.GENERAL INTRODUCTION
2.1 RESEARCH BACKGROUND
2.2 RESEARCH SIGNIFICANCE
2.2.1 Burst release effects and controlled release approach
2.2.2 Biodegradation of silk materials,an important feature to be taken into considerations
2.3 RESEARCH OBJECTIVE
2.4 RESEARCH CONTENT
2.5 WORKFLOW OF THE STUDY
CHAPTER Ⅲ.TRANSGENIC SILKWORMS PRODUCING HUMAN BONE MORPHOGENETIC GROWTH FACTOR2 FUSED TO FIBROIN HEAVY CHAIN VIA MMP SENSITIVE LINKERS
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 Materials
3.2.2 Major instruments and Appliances
3.2.3 General reagents and kits
3.2.4 Media,buffers and solutions setup
3.3 METHODOLOGICAL SECTIONS
3.3.1 Primer design and synthesis
3.3.2 Design and synthesis of hBMP-2 derived genes with MMP linkers at both termini
3.3.3 Plate streaking,bacteria growth and plasmid extraction
3.3.4 Inclusion of MMP sensitive linkers
3.3.5 Construction of the expression cassette and subsequent cloning into piggyBac vector
3.3.6 Construction of recombinant piggyBac vectors
3.3.7 Embryo preparation and microinjection
3.3.8 Embryo development and larvae rearing
3.3.9 Screening and identification of the transgenic silkworms
3.3.10 Validation of transgene integration into silkworm genome
3.3.11 Identification and quantification of recombinant protein from silkworm cocoons
3.4 RESULTS AND DISCUSSIONS
3.4.1 BMP-2 fused to fibroin heavy-chain gene via matrix metalloproteinase sensitive linkers presenting different catalytic activities
3.4.2 Transgenic silkworms expelling BMP-2 as a foreign constituent of the cocoon fiber
3.4.3 Transgenic silkworm lines producing BMP-2 tethered with Matrix metalloproteinase sensitive linkers
3.5 CONCLUSION
CHAPTER Ⅳ.IN VITRO DEGRADATION OF SILK FIBROIN BY MMP-2 ENZYMES SECRETED BY MACROPHAGES
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.2.1 Biological materials
4.2.2 Instrument and equipment
4.2.3 Chemical reagents
4.3 METHODOLOGICAL SECTION
4.3.1 Fabrication of silk based materials
4.3.2 Quantitation of the level of BMP-2 in different silk fibroin materials
4.3.3 Sterilization of silk fibroin slices
4.3.4 Culture of human THP-1 monocyte cells
4.3.5 Differentiation of monocyte THP1 and polarization into pro-inflammatory M1 macrophages
4.3.6 In vitro macrophage-mediated degradation of silk fibroin slices In vitro MMP-mediated degradation system set up
4.3.7 Statistical analysis
4.4 RESULTS
4.4.1 Regeneration processes decreased the content of BMP-2 at certain extent
4.4.2 Macrophages are the prevalent cell type observed on silk fibroin implants and can serve as the source of MMP-2 enzyme
4.4.3 The incorporation of MMP cleavable substrate presenting various kinetic activity into silk fibroin results in different degradation rate
4.4.4 The addition of MMP cleavable peptide promoted the degradation of SF slices
4.5 Conclusions
CHAPTER Ⅴ.IN VITRO MMP-MEDIATED RELEASE AND BIOACTIVITY OF RELEASED BMP-
5.1 INTRODUCTION
5.2 MATERIALS
5.2.1 Cell lines
5.2.2 Major equipment
5.2.3 Reagents and kits
5.3 METHODOLOGICAL SECTION
5.3.1 Generation of silk fibroin aqueous solution
5.3.2 Preparation of macrophage conditioned media:source of MMP-
5.3.3 Ascertain the susceptibility of peptide sites and cleavage by MMP-
5.3.4 In vitro macrophage-mediated release
5.3.5 Quantification of BMP-2 from release media
5.3.6 Quantification of BMP-2 in remained silk fibroin slices
5.3.7 Bioactivity of BMP-2 released from cocoon slices
5.3.8 Statistical analysis
5.4 RESULTS AND DISCUSSIONS
5.4.1 Digestion of cleavable linkers by MMP secreted by macrophages freed BMP-2 out from silk fibroin
5.4.2 The cleavage activity occurred at different rate resulting in tunable release profile
5.4.3 BMP-2 released from transgenic silk fibroin groups stimulated MSC proliferation
5.5 CONCLUSION
CHAPTER Ⅵ.SUMMARY AND CONCLUSIONS
论文创新点
REFERENCES
在读期间发表文章、参加会议及参研课题
ACKNOWLDEMENT
本文编号：3456862
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