特异性结合BMP-2丝素支架的制备及其生物相容性研究

发布时间：2018-05-23 19:43

本文选题：骨形态发生蛋白2 + 特异性结合　；参考：《浙江理工大学》2012年硕士论文

【摘要】：骨形态发生蛋白2（BMP-2）是骨形态发生蛋白家族中的一员，具有极强的诱导成骨能力，，属于转录生长因子β（TGF-β）超家族。通常，该类生长因子在体内的半衰期大约是数秒到数分钟，很容易失去活性。理想的方式是将生长因子与载体材料复合，保持生长因子在体内环境下的生物活性并实现其长期有效释放。而常规的物理吸附方法，存在BMP-2与载体间较低亲和性的问题，导致BMP-2的快速释放。近年来，研究者们尝试对BMP-2与载体的改性等方法来提高二者的亲和性。另一方面，丝素蛋白由于其良好的机械性能、生物相容性等被广泛应用于组织工程、药物释放等领域。本研究采用大肠杆菌BL21原核表达获得人重组BMP-2，系统地研究了人重组BMP-2原核表达纯化和复性，以及复性后蛋白的活性；采用冷冻干燥法制得丝素支架（SF），研究了二种特异性结合BMP-2丝素支架的制备方法，同时分析了其形貌、红外、孔隙率等因素，并考察了二种特异性结合BMP-2丝素支架肝素接枝量，以及对BMP-2吸附和释放行为；然后又分别研究了二种特异性结合支架细胞毒性，负载BMP-2后复合支架的生物相容性。通过原核表达所得到的BMP-2分子量在20kDa左右，与预期一致，在30μg/mL浓度下MG-63细胞的分化，具有良好的生物学活性。冷冻干燥法制得的丝素支架具有多孔结构，孔径分布在100-200μm，孔间连通性较好，红外结果显示其二种特异性结合支架中的SF蛋白结构转变成更加稳定的β折叠结合。HP/SF与HP/L-SF肝素接枝量分别达到了1.80±0.14μg/mg和1.10±0.22μg/mg。体外复合支架对BMP-2的吸附及释放行为研究结果表明，二种特异性结合BMP-2支架HP/SF与HP/L-SF能显著提高BMP-2的吸附量，相对于SF12.21±0.52μg BMP-2的吸附量，HP/SF的吸附量达到了15.15±0.93μg，而HP/L-SF的吸附量则更高，达到了19.65±1.81μg；并且二种支架在有效的抑制了BMP-2突释，相对于SF第一天50.41%的释放，HP/SF与HP/L-SF第一天的释放率分别下降到29.90%、26.82%，到达第七天时，相对于SF78.41%的释放率，HP/SF与HP/L-SF则分别为53.53%、40.97%；细胞结果表明，两种特异性结合BMP-2丝素支架负载BMP-2后均能持续显著地提高MG-63细胞分化水平，而且HP/L-SF组较HP/SF组有更高促进细胞分化的效果。本文针对BMP-2与载体间存在低亲和性，容易快速流失的缺点，采用在丝素支架接枝肝素，从而来提高与BMP-2的亲和性，并成功制备出了二种特异性结合BMP-2丝素支架，负载BMP-2后对MG-63细胞具有良好的生物相容性，是一种良好的BMP-2亲和载体。本研究工作为制备其它新型的BMP-2载体开拓了思路。
[Abstract]:Bone morphogenetic protein 2 (BMP-2), a member of the bone morphogenetic protein family, has a strong induction of osteogenesis and belongs to the transcription factor beta (TGF- beta) superfamily. Usually, the half-life of this growth factor in the body is about several seconds to a few minutes, and it is easy to lose its activity. Ideally, the growth factor and the carrier material are recovered. In addition, the biological activity of growth factors in the body environment is maintained and its long-term effective release is achieved. While the conventional physical adsorption method has the problem of low affinity between BMP-2 and the carrier, resulting in the rapid release of BMP-2. In recent years, researchers have tried to improve the affinity of the two by the modification of BMP-2 and carrier. On the other hand, silk Because of its good mechanical properties and biocompatibility, plain protein has been widely used in tissue engineering, drug delivery and other fields.
In this study, recombinant BMP-2 was obtained by BL21 prokaryotic expression of Escherichia coli. The expression, purification and refolding of recombinant human BMP-2 prokaryotic cells, as well as the activity of recomplex protein were studied. The preparation method of two specific binding BMP-2 silk fibroin scaffolds was studied by freeze drying, and its morphology, infrared and pore were analyzed. Two specific binding heparin grafting amount of BMP-2 silk fibroin scaffold and adsorption and release behavior to BMP-2 were investigated, and the toxicity of two specific binding scaffolds, and the biocompatibility of the composite scaffold after BMP-2 were respectively studied.
The BMP-2 molecular weight obtained through the prokaryotic expression is around 20kDa, and is consistent with the expectation. The differentiation of MG-63 cells at 30 g/mL concentration has good biological activity. The silk fibroin scaffold obtained by freeze drying has a porous structure, the pore size distribution is 100-200 mu m, the inter pore connectivity is better, and the infrared results show the two specific binding scaffolds. The transformation of SF protein structure into a more stable beta folding binding.HP/SF and HP/L-SF heparin reached 1.80 + 0.14 g/mg and 1.10 + 0.22 mu g/mg. in vitro. The adsorption and release behavior of BMP-2 was studied. The results showed that two specific binding BMP-2 scaffolds HP/SF and HP/ L-SF could significantly increase the BMP-2 adsorption capacity, relative to S. The adsorption amount of F12.21 + 0.52 g BMP-2, HP/SF adsorption capacity reached 15.15 + 0.93 mu g, while HP/L-SF adsorption amount was higher, reaching 19.65 + 1.81 mu g, and two kinds of scaffolds were effectively inhibiting the release of BMP-2. Compared with the release of SF first day 50.41%, the release rate of HP/SF and HP/L-SF was reduced to 29.90%, 26.82%, and seventh, respectively. At the day, compared with the SF78.41% release rate, HP/SF and HP/L-SF were 53.53%, 40.97%, respectively. The results showed that the two specific combination of BMP-2 silk fibroin scaffolds could improve the differentiation level of MG-63 cells after BMP-2, and the HP/L-SF group was more effective than the HP/SF group to promote the cell differentiation.
In this paper, in view of the disadvantage of low affinity and easy loss between BMP-2 and carrier, the grafting of heparin on silk fibroin scaffold is used to improve the affinity with BMP-2, and two kinds of specific binding BMP-2 silk fibroin scaffolds have been successfully prepared. After loading BMP-2, the biocompatibility of MG-63 cells is good. It is a good BMP-2 affinity. This work has opened up new ideas for the preparation of other new BMP-2 carriers.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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