本文選題：玉米蛋白　切入點(diǎn)：仿生礦化　出處：《上海交通大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：組織工程在人類生物醫(yī)學(xué)的發(fā)展中起著舉足輕重的作用,而其中骨組織工程支架的制備和細(xì)胞三維培養(yǎng)的微載體技術(shù)扮演著重要的角色。本論文對玉米蛋白材料作為上述組織工程基質(zhì)材料方面的應(yīng)用進(jìn)行了初步探索。首先將玉米蛋白制備成三維(3-D)多孔支架,嘗試采用不同的仿生礦化條件將羥基磷灰石顆粒沉積在多孔支架表面,對其孔隙結(jié)構(gòu)及表面化學(xué)組成等進(jìn)行了表征。通過對3-D多孔支架的仿生礦化條件進(jìn)行改進(jìn),獲得了具有良好孔隙結(jié)構(gòu)、表面均勻沉積了涂層且鈣磷比接近羥基磷灰石的骨組織工程支架。使用環(huán)境友好技術(shù)將玉米蛋白制備成尺寸可控且圓整度高的微載體,評估了細(xì)胞貼壁率、細(xì)胞增殖情況,并與商業(yè)化微載體Cytodex1進(jìn)行對比,發(fā)現(xiàn)細(xì)胞能夠正常粘附并且增殖,細(xì)胞收獲量達(dá)到了商業(yè)微載體水平,為進(jìn)一步應(yīng)用于組織工程提供了新型基質(zhì)材料。方法:用磨具將玉米蛋白制備成5mm×5mm×15mm和Φ2mm×4mm的三維(3-D)多孔支架,并對其外觀形貌和孔隙結(jié)構(gòu)進(jìn)行了表征。將該玉米蛋白多孔支架浸入5倍模擬體液(5×SBF)中,在不同的條件下進(jìn)行仿生礦化。對仿生礦化后的材料也進(jìn)行了孔隙率及表面形貌的表征,并使用能譜儀對羥基磷灰石涂層進(jìn)行了元素分析(EDS)、計(jì)算其鈣磷比,使用X射線衍射儀(XRD)確定涂層的物質(zhì)是羥基磷灰石,通過穿孔實(shí)驗(yàn)評估了羥基磷灰石涂層的牢固程度,為動(dòng)物成骨實(shí)驗(yàn)提供符合條件的樣本。使用環(huán)境友好的甘油熱塑法制備不同尺寸的微載體,并使用熒光顯微鏡觀察所制備微載體的圓整度,測量其直徑并進(jìn)行統(tǒng)計(jì)。通過體外實(shí)驗(yàn)評價(jià)其抗酶解性能。種植Vero細(xì)胞評價(jià)細(xì)胞貼壁和增殖速度并與Cytodex1對比,掃描電子顯微鏡觀察細(xì)胞在微載體上的生長狀態(tài)。結(jié)果與討論:最佳沉積條件為:將三維玉米蛋白多孔支架浸入5倍模擬體液中,置于全溫?fù)u床,在37℃,60rpm轉(zhuǎn)速下仿生礦化3天,在一天半時(shí),更換新鮮的5倍模擬體液,并將多孔支架上下翻轉(zhuǎn)。所獲得的玉米蛋白支架的各個(gè)表面均沉積到相對均勻的羥基磷灰石顆粒,顆粒直徑在2-10μm,各個(gè)面的鈣磷比在1.40-1.67之間。說明仿生礦化效果得到了改進(jìn)。使用甘油作為加熱介質(zhì),將玉米蛋白塑形。得到的微球直徑可控,且分布均勻,圓整度良好。將Vero細(xì)胞種植在微載體表面,結(jié)果顯示細(xì)胞能夠正常貼壁,并能夠增殖,兩者均與商業(yè)廣泛使用的微載體Cytodex1相當(dāng)。說明玉米蛋白具有作為微載體的潛在新用途,不僅可用于疫苗生產(chǎn)目的的動(dòng)物細(xì)胞的大規(guī)模培養(yǎng),而且可拓展至組織工程領(lǐng)域。
[Abstract]:Tissue engineering plays an important role in the development of human beings in biomedical science, including the preparation of bone tissue engineering scaffolds and three-dimensional cell culture micro carrier technology plays an important role. This paper conducts a preliminary exploration of corn protein using the tissue engineering material as matrix materials. The preparation of corn protein the three-dimensional (3-D) porous scaffolds, using biomimetic mineralization conditions of different hydroxyapatite particles deposited on the surface of the porous scaffolds, pore structure and surface chemical composition were characterized. By improving the biomimetic mineralization conditions on 3-D porous scaffolds, obtained has good pore structure, uniform surface coating and deposition of calcium and phosphorus ratio of bone tissue engineering scaffolds. Close to hydroxyapatite using environmentally friendly technology of the corn protein prepared by size controllable and roundness of micro carrier, To evaluate the rate of cell adhesion, cell proliferation, and commercialization of microcarrier Cytodex1 were compared, found that normal cells can adhesion and proliferation, cell harvest reached commercial micro carrier level, provides a new matrix material for further application in tissue engineering. Methods: the three-dimensional corn protein preparation tools into 5mm * 5mm * 15mm * 4mm and 2mm diameter (3-D) porous scaffolds, and the morphology and pore structure were characterized. The corn protein scaffolds immersed in simulated body fluid 5 times (5 * SBF), biomimetic mineralization in different conditions. The biomimetic materials were also carried out after characterization of porosity and surface morphology, and the use of EDS to analyze elements of hydroxyapatite coating (EDS), calculate the ratio of calcium and phosphorus, using X ray diffraction (XRD) coating material is determined by experimental evaluation of hydroxyapatite, perforation of hydroxyl A strong degree of hydroxyapatite coating, to provide qualified samples for experimental animal bone. Microcarrier preparation of different sizes of glycerol heat using environmentally friendly plastic method, and fluorescence microscopy was used to prepare the micro carrier roundness, measure its diameter and statistics. By in vitro experiments to evaluate its anti enzyme properties Vero cell evaluation. Plant cell adherence and proliferation rate and compared with the Cytodex1, the growth state of scanning electron microscopy of cells on microcarrier. Results and conclusion: the best deposition conditions: three-dimensional porous scaffolds immersed in corn protein 5 times simulated body fluid, put the whole temperature shaker, at 37 degrees, 60rpm speed bionic the mineralization of 3 days, in a day and a half, 5 times simulated body fluid replacement of fresh, and the porous scaffold under flip. Each surface corn protein scaffold obtained were deposited onto hydroxyapatite particles relatively uniform, The particle diameter of 2-10 m, each surface of the calcium and phosphorus ratio in the range of 1.40-1.67. The biomimetic mineralization effect is improved. The use of glycerol as the heating medium, the corn protein shaping. Obtained microspheres with controllable diameter, and uniform distribution, good roundness. Vero cells grown on microcarriers. The results showed that the cell can be normal attached to the wall, and is capable of proliferation, microcarrier Cytodex1 both are widely used and quite. Commercial corn protein has potential use as a new micro carrier, not only can be used for vaccine production to large-scale culture of animal cells, and can be extended to the field of tissue engineering.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R318.08

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