三维鱼类胶原支架的制备及其生物相容性研究

发布时间：2018-12-16 00:36

【摘要】：为了评价深海鱼类胶原蛋白用于制备组织工程角膜载体支架的可行性,首次利用鱼皮胶原蛋白进行了三维胶原支架的制备及其生物相容性研究。用源于太平洋鳕鱼(Gadus macrocephalus)的纯化鱼皮胶原蛋白,经冷冻干燥后选用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)/N-羟基丁二酰亚胺(NHS)交联剂制备出三维胶原支架,利用外观照相、光度计透光率、冰冻切片苏木紫-伊红(HE)染色及扫描电镜检测了其透明度和组织结构;在接种Di I(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)荧光标记的非转染人角膜基质(HCS)细胞后,利用冰冻切片HE染色和荧光观察鉴定了细胞的迁入及在支架内的分布情况,生物相容性评价采用MTT(噻唑蓝)和免疫细胞荧光技术检测了支架对HCS细胞活力和标志蛋白及功能蛋白表达的影响。结果显示,所制备的三维胶原支架透明度极高,呈现出均匀的网格状结构,孔径大小为50~130μm;接种HCS细胞并体外培养3 d后,发现有大量细胞迁入支架内且分布较为均匀;支架抽提物对HCS细胞活力没有任何不良影响;且迁入支架内的细胞仍保持有其标志蛋白—波形蛋白,细胞连接形成蛋白—整联蛋白β1、E-钙黏蛋白和间隙连接蛋白-43,膜运输蛋白—钠钾泵,以及抗UV蛋白—乙醛脱氢酶的阳性表达。综上所述,利用太平洋鳕鱼皮胶原蛋白制备的三维胶原支架具有良好的透明度与组织结构,不仅适合HCS细胞的顺利迁入,而且对HCS细胞具有良好的生物相容性,有望作为载体支架用于组织工程角膜的体外构建研究。
[Abstract]:In order to evaluate the feasibility of deep-sea fish collagen used to prepare tissue engineering corneal carrier scaffold, the three-dimensional collagen scaffold was prepared by using fish skin collagen for the first time and its biocompatibility was studied. Purified fish skin collagen derived from Pacific cod (Gadus macrocephalus), Three-dimensional collagen scaffolds were prepared by using 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS) crosslinking agent after freeze-drying. The transparency and structure of frozen section were examined by (HE) staining and scanning electron microscopy. After inoculated with Di I (1 ~ (-1) -dioctadecyl-3 ~ (3 +) -tetramethylindocarbocyanine perchlorate, the migration and distribution of human corneal stromal (HCS) cells were identified by HE staining and fluorescence observation in frozen sections. Biocompatibility was evaluated by MTT (thiazolyl blue) and immunofluorescence technique. The effects of stents on the viability and expression of marker proteins and functional proteins in HCS cells were investigated. The results showed that the three-dimensional collagen scaffold was highly transparent, with a uniform reticular structure with a pore size of 50 ~ 130 渭 m. After inoculating HCS cells and cultured in vitro for 3 days, a large number of cells were found to migrate into the scaffold and distributed evenly. The scaffold extract had no adverse effect on the viability of HCS cells. The cells that migrated into the scaffold still had their marker protein vimentin, cell junction protein-integrin 尾 1-E-cadherin and gap junction protein-43, membrane transport protein-sodium potassium pump. And the positive expression of anti UV protein-acetaldehyde dehydrogenase. In conclusion, the three-dimensional collagen scaffold prepared from Pacific cod skin collagen has good transparency and tissue structure, which is not only suitable for the smooth migration of HCS cells, but also has good biocompatibility for HCS cells. It is expected to be used as carrier scaffold for in vitro construction of tissue engineered cornea.
【作者单位】：中国海洋大学海洋生命学院角膜组织工程重点实验室;
【基金】：国家高技术研究发展计划(863计划)资助项目(2006AA02A132)
【分类号】：R318.08

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