Preparation of lactose-silk fibroin conjugates and their application as a scaffold for hepatocyte attachment
Preparation of lactose-silk fibroin conjugates and their application as a scaffold for hepatocyte attachment
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DOI：
10.1016/s0142-9612(03)00633-1
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发表时间：
2004-03-01
期刊：
影响因子：
14
通讯作者：
Miyashita, T
中科院分区：
文献类型：
--
作者：
Gotoh, Y;Niimi, S;Miyashita, T
我们制备的糖复合物（Lac-CY-SF）的溶解的丝素蛋白（SF）的均相化学修饰与乳糖轴承半乳糖残基使用氰尿酰氯（CY）作为耦合间隔，并检查其应用程序作为肝细胞附着的支架的有用性。通过几个标准评估乳糖共价固定到SF中，包括H-1-NMR测量和与凝集素如蓖麻凝集素（RCA（120））和异硫氰酸荧光素标记的RCA（120）（FITC-RCA（120））的反应。Lac-CY-SF的1H-1-NMR谱显示归因于乳糖的次甲基和亚甲基质子的新的宽峰，并且通过峰的积分强度，确定Lac-CY-SF中乳糖与SF的重量比为0.20。向Lac-CY-SF溶液中加入RCA（120）导致Lac-CY-SF溶液浊度增加。将Lac-CY-SF膜与FITC-RCA（120）孵育，表明Lac-CY-SF膜的荧光从整个表面发射。此外，我们研究了聚苯乙烯培养皿上的Lac-CY-SF缀合物涂层对大鼠肝细胞的附着及其形态的影响。肝细胞在0.1%（w/v）偶联物包被的培养皿上的附着显示出比未包被的培养皿上的附着增加约8倍，与胶原包被的培养皿上的附着相当，而SF包被的培养皿上的附着低于未包被的培养皿上的附着。在偶联物包被的培养皿上培养2.5 h的肝细胞与胶原包被的培养皿上的肝细胞相比显示出较小的圆形形态。在含有100 nM胰岛素和100 nM地塞米松的培养基中培养2天后，缀合物包被的培养皿上的肝细胞形成具有轻微分散形态的单层岛，而在胶原包被的培养皿上培养的肝细胞均匀平铺。这些结果表明，Lac-CY-SF缀合物可用作肝细胞附着的支架，但在缀合物包被的培养皿上培养的肝细胞的形态不同于在胶原包被的培养皿上培养的肝细胞的形态。(C)2003 Elsevier Ltd.保留所有权利。
We prepared glycoconjugates (Lac-CY-SF) by the homogeneous chemical modification of solubilized silk fibroin (SF) with lactose bearing the galactose residue using cyanuric chloride (CY) as a coupling spacer and examined the usefulness of their application as a scaffold for hepatocyte attachment. The covalent immobilization of lactose into SF was assessed by several criteria including H-1-NMR measurements and reactions with lectins such as Ricinus communis agglutinin (RCA(120)) and fluorescein isothiocyanate-labeled RCA(120) (FITC-RCA(120)). The H-1-NMR spectrum of Lac-CY-SF showed new broad peaks attributed to methine and methylene protons of lactose, and by the integrated intensities of the peaks the weight ratio of lactose to SF in Lac-CY-SF was determined to be 0.20. Addition of RCA(120) to Lac-CY-SF solution caused an increase in the turbidity of the Lac-CY-SF solution. Incubation of Lac-CY-SF films with FITC-RCA(120) showed that the fluorescence emitted from the whole surface of Lac-CY-SF films. Furthermore, we examined the effect of the Lac-CY-SF conjugate-coating onto polystyrene culture dishes on the attachment of rat hepatocytes and their morphology. Attachment of hepatocytes onto the 0.1% (w/v) conjugate-coated dishes showed about an 8-fold increase as compared with that on uncoated dishes, being comparable to that on collagen-coated dishes, whereas the attachment on the SF-coated dishes was lower than that on uncoated dishes. Hepatocytes cultured onto the conjugate-coated dishes for 2.5 h showed smaller round-shaped morphology compared to those on collagen-coated dishes. After 2 days of culture in medium containing 100 nM insulin and 100 nM dexamethasone, hepatocytes on the conjugate-coated dishes formed monolayer islands with a slightly dispersed morphology, while hepatocytes cultured on collagen-coated dishes were uniformly spread flat. These results indicate that the Lac-CY-SF conjugates are useful as a scaffold for hepatocyte attachment, but the morphology of hepatocytes cultured on the conjugate-coated dishes is different from that on collagen-coated dishes. (C) 2003 Elsevier Ltd. All rights reserved.