应用4,6-联脒-2-苯基吲哚大体染色法快速观察生物支架材料体外内皮化程度  

Endothelialization of decellularized scaffold evaluated by gross 4, 6-diamidino-2-phenylindole 2hci staining

作　　者：孙洪亮[1] 方宁涛[1] 谢尚喆[1] 高红阳[1] 潘銮凤[1] 

机构地区：[1]复旦大学上海医学院分子生物学实验室,上海市200032

出　　处：《中国临床康复》2006年第25期57-59,共3页Chinese Journal of Clinical Rehabilitation

基　　金：上海市科委基金资助重点项目(04JC14012)~~

摘　　要：目的:建立一种新的形态学方法,以简便快捷地观察生物支架材料体外内皮化的程度。方法:实验于2005-07/2006-02在复旦大学上海医学院分子生物学实验室完成。①新鲜的猪心瓣膜经过十二烷基磺酸钠和脱氧胆酸处理,获得去细胞生物支架。②采用消化法获得脐静脉内皮细胞。③将脐静脉内皮细胞(2×105/cm2)种植于支架上,静态培养10d。用0.5mg/L的4,6-联脒-2-苯基吲哚溶液对再内皮化支架进行直接的大体染色及染色后冰冻切片,同时用苏木精-伊红染色,免疫荧光染色和扫描电镜对支架的内皮化情况进行平行观察。结果:①猪心瓣膜的细胞成分完全脱去,胞外基质保存完好。②原代分离的脐静脉内皮细胞,培养5d后融合成扁平单层,梭形或多角形,具有典型的铺路石样形态;CD31免疫荧光染色细胞表面呈现黄绿色荧光。③脐静脉内皮细胞种植10d后,支架经4,6-联脒-2-苯基吲哚大体染色显示内皮化程度达90%以上,观察结果与电镜观察相同;冰冻切片观察显示支架表面有一融合的内皮细胞单层,与免疫荧光染色及常规苏木精-伊红染色结果相吻合。结论:4,6-联脒-2-苯基吲哚大体染色法可直接观察生物支架再内皮化程度,简便、准确直观,是体外构建组织的一种有效的形态学检测方法。AIM： To develop a new morphological method to conveniently and rapidly observe the endothelialization level of biosynthetic scaffold. METHODS： The experiment was conducted in the Laboratory of Molecular Biology, Shanghai Medical College, Fudan University between July 2005 and February 2006. （1）Fresh porcine heart valves were treated with sodium dodyeypsulphate and deoxyeholate to obtain decellaluized scaffolds. （2）The human umbilical vein endothelial cells （HUVECs） were digested with 0.1% eollagenase A. （3）The HUVECs （2×10^5/cm^2） were seeded on the scaffold and cultured for 10 days. The reendothelialization scaffolds were stained with 0.5 mg/L 4, 6-diamidino-2-phenylindole 2 hci （DAPI） solution and the sections were frozen, and statieally observed by immunostaining, HE staining and scanning electron mieroseope. RESULTS： （1）The cellular components of heart valves were completely removed and the extracellular matrix was well reserved. （2）The HUVECs retained spindled or polygon shaped, with eobblestone appearance after 5 days of euhure; CD31 antigens cells showed green fluorescence. （3）DAPI staining showed that 90% of scaffolds surface was reendothelialized after 10 days of euhure, which was accorded with the observation by scanning electron; The section observation showed the endothelial cells were distributed evenly on the surface and formed a confluent monolayer. The results were accorded with the results of immunostaining and HE staining. CONCLUSION： DAPI staining can conveniently, directly and accurately observe the level of reendothelialization, whieh is an effective morphological method for observation of in vitro construction tissues.

关 键 词：组织工程 心脏瓣膜 支架 

分 类 号：R318[医药卫生—生物医学工程]