硝酸银染色法鉴定生物瓣支架材料体外再内皮化程度的实验研究  

EVALUATION OF RECELLULARIZATION LEVEL OF BIOPROSTHETIC VALVE SCAFFOLD WITH AGNO_3 STAINING TECHNIQUE IN VITRO

作　　者：冯滨 刘迎龙[1] 谢宁[1] 宋来凤[2] 

机构地区：[1]中国医学科学院心血管病研究所中国协和医科大学阜外心血管病医院外科 [2]中国医学科学院心血管病研究所中国协和医科大学阜外心血管病医院病理科,北京100037

出　　处：《中国修复重建外科杂志》2005年第11期910-914,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家高技术研究发展计划(863)基金资助项目(2001AA216061);国家自然科学基金资助项目(30271289);中国博士后科学基金资助项目(2003033224)~~

摘　　要：目的探讨生物瓣支架再内皮化程度的形态学观察方法,为体外构建心脏组织工程瓣提供手段。方法选择经液氮保存的同种主动脉带瓣管道作为生物瓣支架,0.1%十二烷基硫酸钠(sodiumdodecylsulphate,SDS)脱去表面的内皮细胞(endothelialcells,ECs),纤维连接蛋白(bironectin,FN;80μg/ml)预覆盖瓣膜;选择从正常成人骨髓间充质干细胞(mesenchymalstemcells,MSCs)经体外定向诱导分化的ECs作为种子细胞,以>1～2×105个/cm2密度种植于瓣膜支架上,采用含20%胎牛血清和血管内皮细胞生长因子等的DMEM-HG培养基静态培养,0.5%硝酸银染色,于培养的7、14和20d在立体显微镜下观察、摄片,确定其内皮化程度。结果同种生物瓣表面的ECs完全脱去,而细胞外基质成分保存良好;种植细胞与生物瓣支架复合体静态培养的第7、14和20天,其内皮化程度分别为73%、85%和92%。结论硝酸银染色鉴定生物瓣支架再内皮化的方法,简便、经济,是体外构建心脏组织工程瓣形态学观察的一种有效手段。Objective To explore morphological recellularization level of bioprosthetic valve scaffold （BVS） and to provide researching means for fabricating tissue engineered heart valve in vitro. Methods The homograft bioprosthetic aortic tube valve was selected as BVS, which was conserved by liquid nitrogen, and its endothelial cells （ECs） were removed by 0. 1% sodium dodecylsulphate （SDS）. As implantation cells, the endothelial cells （ECs） differentiating from human bone marrow mesenchymal stem cells （MS（Ts） in vitro were implanted with high-density seeding （〉10^3cells/cm^2） on the BVS, which was covered by fibronectin （80μg/ml） in advance. The complex structure was statically cultured in I）MEM （high glucose） with 20% FBS and VEGF （10 ng/ml） for about 20 days in vitro and stained by 0. 5% AgNO3. The morphological structure was observed and photographed by stereomicroscope to detect the reecllularization level. Results The ECs of the bioprosthetic valve were not only removed completely, but also the collagen fiber and elastic fibers were reserved. The ECs differentiating from MSCs were successfully implanted on the HBS, whose recellularization levels on 7th, 14th and 20th day were 73%, 85%, and 92% respectively. Conclusion AgNO3 staining technique is effective, convenient, and economic in evaluating the recellularization level of BVS. It is an effective method in morphological observation for fabricating tissue engineered heart valve in vitro.

关 键 词：组织工程瓣膜 硝酸银染色 生物瓣支架 内皮细胞 

分 类 号：R318.08[医药卫生—生物医学工程] R654.2[医药卫生—基础医学]