具有良好细胞相容性的脱细胞血管支架的制备  

Preparation of decelluarized vascular scaffold with perfect biocompatibility

作　　者：方宁涛[1] 潘銮凤[1] 谢尚喆[1] 吴春根[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200032

出　　处：《复旦学报（医学版）》2006年第4期547-551,共5页Fudan University Journal of Medical Sciences

基　　金：上海市科委基金资助重点项目(04JC14012);复旦大学Med-X基金资助项目

摘　　要：目的制备具有良好细胞相容性的脱细胞血管支架材料,为构建组织工程血管奠定基础。方法用含有脱氧胆酸钠、SDS、EDTA的低渗液和等渗液以及核酸酶对猪的胸主动脉进行脱细胞处理,使用HE、Movat五色法、DAPI染色及电镜观察处理结果。以MTT法分析脱细胞溶液的细胞毒性,通过种植内皮和平滑肌细胞,分析支架的细胞相容性。结果上述方法使用的脱细胞溶液细胞毒性低;经该法处理后,血管的细胞成分完全去除,胶原纤维、弹性纤维、糖蛋白等主要基质成分充分保留,组织结构完整,内皮细胞和平滑肌细胞可以在上面黏附生长,形成完整的细胞层。结论利用脱氧胆酸钠、SDS、EDTA和核酸酶联合消化的方法,可制备出基质成分充分保留、细胞相容性优良的脱细胞血管支架,适于构建组织工程血管。Purpose To prepare decelluarized vascular scaffold with excellent biocompatibility for vessel tissue engineering. Methods Fresh porcine aortic vessels were decellularized by using a 3-step hypotonic-detergents-enzymatic extraction method involving sodium dodecyl sulfate （ SDS ）, sodium deoxycholate （DOC） and ethylenediaminetetraacetic acid （EDTA） in combination with RNase and DNase. The results were examined by the HE stain, Movate-pentachrome stain, DAPI stain and transmission electron microscopy. The cytotoxicity of 3 detergent solutions was determined by MTT assay. Human umbilical vein endothelial cells （ HUVECs ） and canine smooth muscle cells （ SMCs ） were seeded onto the luminal surface of scaffolds and then examined by HE stain, immunostains and scanning electron microscopy. Results After treated by 0.01% SDS, 0.25 % sodium deoxycholate and 0. 1% EDTA, the vessels were stained by HE and DAPI method which showed that cells were completely removed from and DNA was not detected in the entire aortic conduits. Movat-pentachrome stain showed the extracellular matrix structures and main components such as collagen, elastin and glycosaminoglycans were well preserved. These facts were further verified by transmission electron microscopy. MTT assay revealed the low cytotoxicity of the detergent solution used in this method. The seeded HUVECs and SMCs had showed the potentials of spread and proliferation on the luminal surface of decellularized vascular scaffold, and the confluent endothelial cells could be observed by scanning electron microscopy. Conclusions By Hypotonic-detergents-enzymatic extraction, the excellent vessel scaffold with well biocompatibility and main components preserved was made, and suitable for vessel tissue engineering application.

关 键 词：血管组织工程 脱细胞血管基质 生物材料 生物相容性 

分 类 号：R318.11[医药卫生—生物医学工程]