骨髓间充质干细胞来源的内皮细胞构建组织工程瓣膜及其抗血栓作用  

Bone mesenchymal stem cell derived endothelial cells for constructing tissue engineered heart valve and its anti-thrombotic effects

作　　者：方宁涛[1] 张宇浩[2] 孙洪亮[1] 谢尚喆[1] 高红阳[1] 裴鹏[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 [2]复旦大学附属中山医院神经内科,上海市200032

出　　处：《中国临床康复》2006年第45期27-29,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:分析骨髓间充质干细胞来源的内皮细胞构建组织工程瓣膜的可行性,评价其抗血栓功能。方法:实验于2005-08/2006-04在复旦大学上海医学院分子生物学实验室完成。①人骨髓来源为中山医院胸外科手术摘除的一段肋骨(患者知情同意);取健康志愿者静脉血进行血小板黏附观察;新鲜猪心瓣膜取自上海复新屠宰场。②利用胰酶-乙二胺四乙酸、核酸酶处理猪心瓣膜制备脱细胞支架。将摘除的一段肋骨无菌条件下用含肝素的磷酸盐缓冲液充分冲洗骨髓腔,进行骨髓间充质干细胞的分离培养及体外诱导分化。③经体外诱导成的内皮细胞后,种植于瓣膜支架,构建组织工程瓣膜。体外培养1周后,取组织工程瓣膜及未种细胞的空支架做血小板黏附试验。以CD31、vWF对细胞进行鉴定,采用苏木精-伊红染色、免疫荧光及扫描电镜对组织工程瓣膜进行评价。结果:①骨髓间充质干细胞定向诱导为内皮细胞及其性质鉴定:骨髓间充质干细胞定向诱导为内皮细胞后,CD31及vWF染色由阴性转为阳性,呈现出“铺路石”样形态。②瓣膜组织形态观察结果:猪心瓣膜的细胞成分完全去除,胞外基质保存良好,组织工程瓣膜表面形成一连续的内皮细胞单层。③血小板黏附情况:扫描电镜显示空支架表面有大量血小板聚集和黏附,而组织工程瓣膜表面无此现象,显示了良好的抗血栓性能。结论:骨髓间充质干细胞来源的内皮细胞可以用来构建具有良好的抗血栓性能组织工程瓣膜。AIM： To explore the feasibility of constructing tissue engineered heart valve with bone mesenchymal stem cell （BMSC） derived endothelial cells, and test their anti-thrombotic potentials. METHODS： The experiment was conducted in the Laboratory of Molecular Biology, Shanghai Medical College, Fudan University between August 2005 and April 2006. ①Human bone marrow was from a section of ribs （the patient was informed and agreed） removed in chest surgery operation of Zhongshan Hospital; the venous blood of healthy volunteers was taken for platelet adhesion test; the fresh porcine heart valves were from Shanghai Fuxin Slaughter House. ② Decellularized porcine heart valves were prepared by using Trypsin-ethylenediamine tetraacetic acid and DNase. The cavitas medullaris of the removed rib was washed sterilely with phosphate buffer solution containing calparine to isolate and culture the BMSCs and induce the differentiation in vitro. ③The endothelial cells differentiated in vitro were seeded onto the surface of decellularized scaffolds to construct tissue engineered heart valve. After cultured for 1 week, the tissue engineered heart valve and unseeded scaffold were tested by platelet adhesion experiments. Cells were identified with CD31 and vWF; tissue engineered heart valve were examined by HE staining, immunostains and scanning electron microscopy. RESULTS： ①Identification of endothelial ceils induced by BMSCs： BMSCs derived endothelial cells were positively stained for CD31 and vWF and displayed cobblestone-like morphologic characteristics. ② Morphological observation of valves： All cells of decellularized heart valves were removed and extracellular matrix was well protected; an uninterrupted endothelial monolayer was formed on the surface of tissue engineered heart valve. ③Platelet adhesion： Scanning electron microscopy showed that there were platelets aggregation and adhesion on the surface of unseeded scaffold, which was not found on tissue engineered heart valve, displaying a good ant

关 键 词：心脏瓣膜 组织工程 骨髓干细胞 间质干细胞 内皮细胞 血栓形成/治疗 

分 类 号：R394.2[医药卫生—医学遗传学]