鳖源胶原蛋白提取纯化及在生物材料中的应用

Isolation, purification of collagen from soft-shelled turtle calipash for application in biomaterial

摘要:

近年来，胶原蛋白因其良好的生物学性能在生物材料应用中得到越来越多的关注，为了建立一种快速高效的鳖源胶原蛋白纯化方法和探究其在生物材料中的应用价值，首先用Van Gieson 染色法和苦味酸-天狼星红染色法观察裙边胶原纤维组织结构，发现裙边胶原纤维含量非常高且类型主要是Ⅰ型。采用不同截留分子量的透析袋对裙边胶原蛋白粗提液进行直接透析纯化，发现截留分子量为100 kDa的透析袋在透析48 h后对裙边胶原蛋白的纯化效果最好，SDS-PAGE检验显示几乎没有杂带。对裙边胶原蛋白生物学性能包括吸水性、体外降解性进行考察，其吸水力和持水力分别高达12.06 g/g和98.21%，且在72 h后被完全降解；对裙边胶原蛋白海绵的溶血性、皮肤致敏性、肝部创伤止血性、促创伤皮肤愈合性进行了研究，并和交联胶原进行比较，发现裙边胶原蛋白和交联胶原均不会造成SD大鼠溶血和皮肤过敏，二者均具有良好的止血效果，且裙边胶原蛋白能显著缩短创伤皮肤愈合时间，具有良好的促创伤皮肤愈合性，而交联胶原效果欠佳。本研究表明裙边胶原蛋白表现出了优良的生物学性能，在生物材料领域具有很大应用价值。

Abstract:

Recently, research on collagen attracts more interests due to its good biological compatibility. The present study attempted to establish a fast and efficient method to purify collagen from soft-shelled turtle and to explore its application in biological materials. The structure and type of collagen fiber in calipash were determined by van Gieson staining and Picrosirius red staining, which could contribute to the isolation of collagen from soft-shelled turtle Calipash (STCC). Collagen fibers were in high content and the main collagen fiber was type I in STCC. The crude STCC solution was purified by dialysis with different cut-off molecular weight. SDS-PAGE demonstrated that the best purification was in applying 100 kDa dialysis bags after 48 h. The water absorbing capacity and holding capacity of STCC were up to 12.06 g/g and 98.21%, respectively. STCC can be degraded by collagenase in vitro entirely after 72 h. The hemolysis, skin sensitization, hemostatic and wound healing of STCC were determined by using SD rat model, and the collagen cross-linked by glutaric dialdehyde was set as a comparison. STCC and STCC cross-linked did not result in destructed red blood cell, inflamed and sensitized skin. Both materials exhibited good hemostatic effect. Thus, STCC improved the wound healing efficiently. This study implies a potential of STCC in the field of biomaterial.