中文摘要：

目的：应用动物来源的细胞构建组织工程化心脏瓣膜的研究较多,本实验特征在于观察人骨髓间质干细胞与脱细胞猪主动脉瓣膜支架体外构建组织工程心脏瓣膜,并探讨其作为种子细胞的可行性。 方法：实验于2005-12/2006-10在解放军第二军医大学长海医院全军胸心外科研究所完成,研究方案获伦理委员会批准。①实验材料：取开胸术中切除的肋骨骨髓,术前已经患者知情同意。新鲜猪心脏瓣膜取自上海复新屠宰场。②实验方法：Ficoll密度梯度离心从肋骨骨髓中获取骨髓单个核细胞,体外扩增培养后鉴定。采用去污剂和酶消化法将猪主动脉瓣叶行脱细胞处理,种植人骨髓间质干细胞。③实验评估：采用EnVision二步法染色鉴定人骨髓间质干细胞形态及细胞表型;采用苏木精-伊红染色、维多利亚蓝染色和Van Gieson氏染色法观察细胞在脱细胞猪主动脉瓣叶支架上的生长特点;扫描电镜观察静态构建组织工程心脏瓣膜的形态。 结果：①人骨髓间质干细胞形态及细胞表型鉴定：人骨髓间质干细胞为梭形,抗平滑肌抗体和波形蛋白染色阳性,CD31及Ⅷ因子染色阴性。②脱细胞猪主动脉瓣膜支架的组织学观察：猪去细胞瓣膜支架去细胞完全,弹力纤维及胶原纤维保持完整,种植的人骨髓间质干细胞在脱细胞瓣叶表面形成完整的细胞层。③构建组织工程心脏瓣膜的形态学观察：扫描电镜示瓣膜表面细胞层完整,细胞呈梭形复层生长。 结论：人骨髓间质干细胞体外具有自然分化为肌成纤维细胞的特性,在去细胞猪主动脉瓣膜上生长良好,可作为一种有前途的种子细胞构建组织工程心脏瓣膜。

英文摘要：

AIM： Studies on tissue engineered heart valve （TEHV） constructed by animal-derived cells are many. This experiment was designed to observe the TEHV constructed by human bone marrow mesenchymal stem cells （hBMSCs） and deceUularized porcine aortic valve matrix in vitro and explore the feasibility as seed cells. METHODS： The experiment was carried out in Institute of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University of Chinese PLA from December 2005 to October 2006. ①Human bone marrow was collected from the ribs excised in thoracotomy after the patients were informed and agreed. The fresh pomine heart valves were obtained from Shanghai Fuxin Slaughter House. ②Bone marrow mononuclear cells （BMNCs） were harvested from ribs by Ficoll density gradient centrifugation, cultured and amplified in vitro and then identified. Decellularized porcine aortic valves were treated with detergent and enzymatic procedure. The hBMSCs were seeded on the decellularized porcine aortic valve matrix in vitro.③Cellular morphology and phenotype of hBMSCs were determined by EnVision two-step staining. The growth characteristics of hBMSCs on decellularized porcine aortic valves were determined by haematoxylin-eosin （HE） staining, Victoria blue （VB） staining and Van Gieson （VG） staining and the morphology of constructed TEHV was evaluated by scanning electron microscopy （SEM）. RESULTS： ① Morphology and phenotype of hBMSCs： The hBMSCs were spindle-shaped with positive staining of anti-smooth muscle antibody （a-SMA） and vimentin and negative staining of CD31 and Ⅷ factor. ② Histological observation of the deceUularized porcine aortic valves： The decellularized procedure resulted in complete removal of the cellular components while the elastic fibers and collagenous fibers were well preserved. The hBMSCs seeded on the matrix formed a continuous multilayer on the surface of TEHV. ③Morphology observation of the constructed TEHV： Scanning electron microscope （S