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  本文選題：骨形態(tài)發(fā)生蛋白2 + 特異性結合　； 參考：《浙江理工大學》2012年碩士論文

【摘要】：骨形態(tài)發(fā)生蛋白2（BMP-2）是骨形態(tài)發(fā)生蛋白家族中的一員，具有極強的誘導成骨能力，，屬于轉錄生長因子β（TGF-β）超家族。通常，該類生長因子在體內(nèi)的半衰期大約是數(shù)秒到數(shù)分鐘，很容易失去活性。理想的方式是將生長因子與載體材料復合，保持生長因子在體內(nèi)環(huán)境下的生物活性并實現(xiàn)其長期有效釋放。而常規(guī)的物理吸附方法，存在BMP-2與載體間較低親和性的問題，導致BMP-2的快速釋放。近年來，研究者們嘗試對BMP-2與載體的改性等方法來提高二者的親和性。另一方面，絲素蛋白由于其良好的機械性能、生物相容性等被廣泛應用于組織工程、藥物釋放等領域。 本研究采用大腸桿菌BL21原核表達獲得人重組BMP-2，系統(tǒng)地研究了人重組BMP-2原核表達純化和復性，以及復性后蛋白的活性；采用冷凍干燥法制得絲素支架（SF），研究了二種特異性結合BMP-2絲素支架的制備方法，同時分析了其形貌、紅外、孔隙率等因素，并考察了二種特異性結合BMP-2絲素支架肝素接枝量，以及對BMP-2吸附和釋放行為；然后又分別研究了二種特異性結合支架細胞毒性，負載BMP-2后復合支架的生物相容性。 通過原核表達所得到的BMP-2分子量在20kDa左右，與預期一致，在30μg/mL濃度下MG-63細胞的分化，具有良好的生物學活性。冷凍干燥法制得的絲素支架具有多孔結構，孔徑分布在100-200μm，孔間連通性較好，紅外結果顯示其二種特異性結合支架中的SF蛋白結構轉變成更加穩(wěn)定的β折疊結合。HP/SF與HP/L-SF肝素接枝量分別達到了1.80±0.14μg/mg和1.10±0.22μg/mg。體外復合支架對BMP-2的吸附及釋放行為研究結果表明，二種特異性結合BMP-2支架HP/SF與HP/L-SF能顯著提高BMP-2的吸附量，相對于SF12.21±0.52μg BMP-2的吸附量，HP/SF的吸附量達到了15.15±0.93μg，而HP/L-SF的吸附量則更高，達到了19.65±1.81μg；并且二種支架在有效的抑制了BMP-2突釋，相對于SF第一天50.41%的釋放，HP/SF與HP/L-SF第一天的釋放率分別下降到29.90%、26.82%，到達第七天時，相對于SF78.41%的釋放率，HP/SF與HP/L-SF則分別為53.53%、40.97%；細胞結果表明，兩種特異性結合BMP-2絲素支架負載BMP-2后均能持續(xù)顯著地提高MG-63細胞分化水平，而且HP/L-SF組較HP/SF組有更高促進細胞分化的效果。 本文針對BMP-2與載體間存在低親和性，容易快速流失的缺點，采用在絲素支架接枝肝素，從而來提高與BMP-2的親和性，并成功制備出了二種特異性結合BMP-2絲素支架，負載BMP-2后對MG-63細胞具有良好的生物相容性，是一種良好的BMP-2親和載體。本研究工作為制備其它新型的BMP-2載體開拓了思路。
[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.
【學位授予單位】：浙江理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R318.08

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