RGD-重组蛛丝蛋白和柞蚕丝素蛋白作为组织工程支架材料的研究
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摘要
蜘蛛丝蛋白纤维具有较高的强度、弹性、柔韧性、伸长度以及良好的生物降解性,在组织工程支架材料领域引起了极大的研究兴趣。RGD(Arg-Gly-Asp)三肽序列是细胞表面整合素的特异配体,能够促进细胞的黏附。本室建立了利用基因工程和高密度发酵技术生产RGD-重组蜘蛛蛋白以及重组蛋白规模纯化的工艺,为其后续研究奠定了基础。柞蚕丝是以野柞蚕茧为原料缫制而成的天然蚕丝纤维,是我国的宝贵资源,但目前主要用作普通的纺织原料。柞蚕丝可以编织成形状类似肌腱的编织带,同时,其优异的力学性能使其能够承受机体的拉伸应力。此外,柞蚕丝中还含有天然存在的RGD序列。因此,柞蚕丝纤维作为一种潜在的组织工程肌腱支架材料,有着广泛的应用前景。
本研究制备了两种RGD蛋白(重组蛛丝蛋白pNSR-16和柞蚕丝素蛋白)的多孔复合支架材料,并首次利用静电纺丝技术制备了pNSR-16纳米复合支架材料;通过傅立叶转换红外光谱(FTIR)和力学性能的测定,对制膜过程中两种蛋白分子构象或复合材料力学性能的影响因素进行了探讨,结果表明:甲酸能促进pNSR-16分子构象向无规卷曲转变,溴化锂、醇类、高分子材料(聚乙烯醇和壳聚糖)可以诱导pNSR-16分子构象向β-折叠转化;小分子增塑剂、高分子量PVA能够促进柞蚕丝素蛋白分子构象向β-折叠转变,提高加热温度、交联、增塑剂PEG则破坏了其β-折叠构象,高分子量PVA、甘油增塑、化学交联、75%乙醇变性均能提高柞蚕丝素蛋白复合材料的力学性能。
通过浸提液毒性实验和细胞与支架的体外联合培养,考察了两种蛋白支架材料的细胞相容性。根据细胞毒性实验ISO10993制定的评价标准得出,两种支架材料的浸提液毒性为1级,反应合格。扫描电镜观察结果表明两种支架均能促进NIH3T3细胞的黏附和生长,具有良好的细胞相容性。
分别利用肌腱细胞和兔跟腱损伤模型,从体外和体内两个水平对柞蚕丝纤维作为组织工程肌腱支架材料的可行性进行了研究。细胞与支架的体外联合培养结果表明:柞蚕丝纤维能够支持肌腱细胞的黏附和生长。体内实验通过大体观察、组织学染色、免疫组化、扫描电镜观察以及生物力学测定等结果表明,柞蚕丝能够支持兔跟腱的修复,初步认为其作为组织工程肌腱支架材料具有一定的可行性。
Owning to its excellent strength, elasticity, flexibility, elongation and biodegradability, spider silk has caused great attention in tissue engineering scaffold material field. RGD (Arg-Gly-Asp) tripeptide sequence, which can promote cell adhesion, is the specific ligand to the integrin on the cell surface. Our lab staff has set up a method to prepare recombinant spider silk by gene engineering and high density fermentation technique. Antheraea pernyi silk is produced by wild A. pernyi silkworm cocoon. It is a precious resource in China. However, it is noly used as a raw material of textile industry presently. Reports on its application in tissue engineering scaffold were scarce. With its RGD tripeptide sequence, to be richly endowed by nature, A. pernyi silk fibroin has superiority in tissue engineering scaffold material field. A. pernyi silk fibroin can be knitted to a tendon-shaped braid, and it can also endure the elongation stress of the body with its outstanding mechanical properties. Therefore, the application of A. pernyi silk fibroin as tissue engineering tendon scaffold has a widely perspective.
In this research, porous complex scaffolds of the two RGD-containing proteins (recombinant spider silk fibroin pNSR-16 and A. pernyi silk fibroin) were prepared. As a first, we obtained recombinant spider silk fibroin complex nanofiber scafflod by electrospinning. Application of Fourie transform infrared spectroscopy (FTIR) and mechanical property determination, the influencing factors to the protein molecular conformation and mechanical properties of the complex scaffold were discussed. Results showed that Formyl Acid promote the transformation of pNSR-16 to random coil, while LiBr, heating, denaturation and macromolecule material (PVA and CS) promte the transformation toβ-fold. It was also found that micromolecule elasticizer and PVA with high molecular weight promote the transformation toβ-fold of A. pernyi silk fibroin, while raising heating temperature, crosslinking and PEG destroy theβ-fold conformation of A. pernyi silk fibroin. According to the differences of mechanical properties between different materials, it can be concluded that PVA with high molecular weight, elasticizer glycerol, chemical crosslinking and denaturation by 75% ethyl alocohol enhance the mechanical properties of A. pernyi silk fibroin complex material.
Cyto-compatibility of the porous complex scaffold of the two RGD-containing proteins was investigated by cytotoxicity determination of the extract of the scaffolds and preparation of Cell-scaffold constructs. According to the evaluation criterion in ISO10993, cytotoxicity of the extract of the two scaffolds is of ranking I and qualified. Results showed that both the scaffolds promote the adhesion and propagation of NIH3T3, namely, both the scaffolds have good cyto-compatibility.
The feasibility of A. pernyi silk fibroin to be used as tissue engineering tendon scaffold was investigated in vitro and in vivo, respectively, utilizing tenocytes and tendon injury rabbit model. In vitro results showed that A. pernyi silk fibroin promote the adhesion and propagation of the tenocytes. Results on macrographic examination, histological examination, immunohistochemical examination, scanning electron micrograph examination and biomechanical determination reveled that A. pernyi silk fibroin promote the recovery of the injury tendon of the rabbit in vivo. Prelimanary, we conclude that the application of A. pernyi silk fibroin as tissue engineering tendon scaffold is feasible.
本研究制备了两种RGD蛋白(重组蛛丝蛋白pNSR-16和柞蚕丝素蛋白)的多孔复合支架材料,并首次利用静电纺丝技术制备了pNSR-16纳米复合支架材料;通过傅立叶转换红外光谱(FTIR)和力学性能的测定,对制膜过程中两种蛋白分子构象或复合材料力学性能的影响因素进行了探讨,结果表明:甲酸能促进pNSR-16分子构象向无规卷曲转变,溴化锂、醇类、高分子材料(聚乙烯醇和壳聚糖)可以诱导pNSR-16分子构象向β-折叠转化;小分子增塑剂、高分子量PVA能够促进柞蚕丝素蛋白分子构象向β-折叠转变,提高加热温度、交联、增塑剂PEG则破坏了其β-折叠构象,高分子量PVA、甘油增塑、化学交联、75%乙醇变性均能提高柞蚕丝素蛋白复合材料的力学性能。
通过浸提液毒性实验和细胞与支架的体外联合培养,考察了两种蛋白支架材料的细胞相容性。根据细胞毒性实验ISO10993制定的评价标准得出,两种支架材料的浸提液毒性为1级,反应合格。扫描电镜观察结果表明两种支架均能促进NIH3T3细胞的黏附和生长,具有良好的细胞相容性。
分别利用肌腱细胞和兔跟腱损伤模型,从体外和体内两个水平对柞蚕丝纤维作为组织工程肌腱支架材料的可行性进行了研究。细胞与支架的体外联合培养结果表明:柞蚕丝纤维能够支持肌腱细胞的黏附和生长。体内实验通过大体观察、组织学染色、免疫组化、扫描电镜观察以及生物力学测定等结果表明,柞蚕丝能够支持兔跟腱的修复,初步认为其作为组织工程肌腱支架材料具有一定的可行性。
Owning to its excellent strength, elasticity, flexibility, elongation and biodegradability, spider silk has caused great attention in tissue engineering scaffold material field. RGD (Arg-Gly-Asp) tripeptide sequence, which can promote cell adhesion, is the specific ligand to the integrin on the cell surface. Our lab staff has set up a method to prepare recombinant spider silk by gene engineering and high density fermentation technique. Antheraea pernyi silk is produced by wild A. pernyi silkworm cocoon. It is a precious resource in China. However, it is noly used as a raw material of textile industry presently. Reports on its application in tissue engineering scaffold were scarce. With its RGD tripeptide sequence, to be richly endowed by nature, A. pernyi silk fibroin has superiority in tissue engineering scaffold material field. A. pernyi silk fibroin can be knitted to a tendon-shaped braid, and it can also endure the elongation stress of the body with its outstanding mechanical properties. Therefore, the application of A. pernyi silk fibroin as tissue engineering tendon scaffold has a widely perspective.
In this research, porous complex scaffolds of the two RGD-containing proteins (recombinant spider silk fibroin pNSR-16 and A. pernyi silk fibroin) were prepared. As a first, we obtained recombinant spider silk fibroin complex nanofiber scafflod by electrospinning. Application of Fourie transform infrared spectroscopy (FTIR) and mechanical property determination, the influencing factors to the protein molecular conformation and mechanical properties of the complex scaffold were discussed. Results showed that Formyl Acid promote the transformation of pNSR-16 to random coil, while LiBr, heating, denaturation and macromolecule material (PVA and CS) promte the transformation toβ-fold. It was also found that micromolecule elasticizer and PVA with high molecular weight promote the transformation toβ-fold of A. pernyi silk fibroin, while raising heating temperature, crosslinking and PEG destroy theβ-fold conformation of A. pernyi silk fibroin. According to the differences of mechanical properties between different materials, it can be concluded that PVA with high molecular weight, elasticizer glycerol, chemical crosslinking and denaturation by 75% ethyl alocohol enhance the mechanical properties of A. pernyi silk fibroin complex material.
Cyto-compatibility of the porous complex scaffold of the two RGD-containing proteins was investigated by cytotoxicity determination of the extract of the scaffolds and preparation of Cell-scaffold constructs. According to the evaluation criterion in ISO10993, cytotoxicity of the extract of the two scaffolds is of ranking I and qualified. Results showed that both the scaffolds promote the adhesion and propagation of NIH3T3, namely, both the scaffolds have good cyto-compatibility.
The feasibility of A. pernyi silk fibroin to be used as tissue engineering tendon scaffold was investigated in vitro and in vivo, respectively, utilizing tenocytes and tendon injury rabbit model. In vitro results showed that A. pernyi silk fibroin promote the adhesion and propagation of the tenocytes. Results on macrographic examination, histological examination, immunohistochemical examination, scanning electron micrograph examination and biomechanical determination reveled that A. pernyi silk fibroin promote the recovery of the injury tendon of the rabbit in vivo. Prelimanary, we conclude that the application of A. pernyi silk fibroin as tissue engineering tendon scaffold is feasible.
引文
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