蠶絲作為一種來源于家蠶（Bombyx mori）的天然高分子材料,以其優(yōu)異的性能而聞名,并因其作為可應(yīng)用于骨組織工程的生物材料而受到人們的關(guān)注。然而天然蠶絲材料尚不能滿足骨組織工程生物材料某些特定的需求,例如天然絲材料缺乏骨相關(guān)生長因子與合適降解速率。骨形態(tài)發(fā)生蛋白（Bone Morphogenetic Protein,BMP-2）是骨組織再生過程中不可或缺的生長因子,其已經(jīng)被廣泛應(yīng)用于蠶絲生物材料研究領(lǐng)域。迄今為止,不同的偶聯(lián)方案已被用于賦予多孔3D海綿、膜、微粒和電紡絲墊等絲素創(chuàng)制材料新功能和能夠釋放出BMP-2。絲素生物材料與BMP-2的偶聯(lián)多采用非共價(jià)偶聯(lián)的策略,這種結(jié)合方式在生物材料使用的最初幾天易引起B(yǎng)MP-2發(fā)生“突釋”現(xiàn)象。因此,如何能夠在賦予蠶絲生物材料新功能情況下并能夠控制功能蛋白或因子的釋放成為這類蠶絲材料應(yīng)用研究的熱點(diǎn)和難點(diǎn)。雖然通過直接添食、化學(xué)改性、中尺度組裝和大尺度混合等改性方法,能夠?qū)崿F(xiàn)賦予蠶絲材料新的所需功能,但是這些策略需要每次使用時(shí)對(duì)材料進(jìn)行復(fù)雜的改性實(shí)驗(yàn)而且很難保證獲得的每批材料的改性一致性。家蠶種系轉(zhuǎn)化（germline transformat... 

【文章來源】：西南大學(xué)重慶市 211工程院校 教育部直屬院校

【文章頁數(shù)】：135 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
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
論文創(chuàng)新點(diǎn)
REFERENCES
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