经液氮保存具有活性的同种生物瓣脱细胞支架的制备  

Preparation of Decellularized Scaffold of Active Homograft Bioprosthetic Valve Preserved by Liquid Nitrogen

作　　者：冯滨[1] 刘迎龙[1] 谢宁[1] 阮英卯[1] 徐新林[1] 宋来凤[1] 

机构地区：[1]中国医学科学院中国协和医科大学阜外心血管病医院外科,北京100037

出　　处：《生物医学工程与临床》2004年第3期125-128,F002,共5页Biomedical Engineering and Clinical Medicine

基　　金：国家863高技术研究发展计划资助项目(No.2001AA216061);国家自然科学基金资助项目(No.30271289);中国博士后科学基金(No.2003033224)

摘　　要：目的为体外构建组织工程瓣提供脱细胞的同种生物瓣支架材料,并建立相应的技术方法。方法选取经液氮保存、具有活性的同种生物带瓣管道作为实验材料,用含十二烷基硫酸钠(SDS)的Tris低渗缓冲液脱去同种带瓣管道存在的细胞;固定剂固定后,分别行HE染色、胶原纤维和弹力纤维染色的光镜和扫描电镜观察、摄片。结果用质量浓度为0.03%SDS的Tris低渗缓冲液与同种瓣膜共同孵育48h,完全脱去了表面的内皮细胞(ECs),又较完整地保留了胶原纤维和弹力纤维等细胞外基质主要成分,其形态学结构在脱细胞前后无明显改变;瓣叶中心部位有部分成纤维细胞存留。而对于脱主动脉壁细胞,质量浓度为0.1%SDS更合适。结论质量浓度为0.03%～0.1%SDS的低渗缓冲液对制备脱细胞的同种生物瓣支架效果较佳,有利于同种瓣再内皮化时种植细胞的粘附和扩增,可进一步应用于组织工程瓣体外构建的研究。Objective To provide the scaffold material of decellularized homograft bioprosthetic valve (HBV)for establishing of tissue engineering cardiac valve in vitro,and related technique. Methods The active homograft bioprosthetic pediculated conduit preserved in liquid nitrogen were selected as experimental material and decellularized by sodium dodecyl sulplate(SDS) containing low osmotic Tris buffer. After fixing it with fixative,stained by hematoxylin and eosin(HE) and special staining for elastic fiber and collagen fibers. Then the material was observed and photographed by light microscope and scanning electron microscope (SEM). Results The endothelial cells of HBV were removed completely when the leaflets of HBV were incubated with 0.03 % SDS containing Tris buffer for 48 hours. The main components of extracellular matrix in their collagen and elastic fiber were reserved. There was no significant difference in morphological structure of HBV between pre and post decellularized. The concentration of SDS should increase to 0.1 % for decellularization of aortic wall. Conclusion The better result will be obtained in HBV decellularization with concentration of 0.03 %～0.1 % SDS containing low osmotic Tris buffer,it is beneficial for adhesion and amplification of implanted cells in HBV reendothelialization. This technique can further be applied in the study on tissue engineering construction of bioprosthetic valve in vitro.

关 键 词：组织工程瓣 脱细胞 细胞洗涤剂 支架材料 同种生物瓣 

分 类 号：R318.08[医药卫生—生物医学工程]