人细胞外基质包被猪去细胞瓣膜支架构建的组织工程心脏瓣膜**☆

背景：去细胞处理的猪主动脉瓣膜植入人体后发生了免疫排斥反应，分析引起免疫排斥反应的原因是由于猪瓣膜的细胞外基质与宿主发生接触引起了免疫激活。 目的：试图将人自身的细胞外基质履盖去细胞猪瓣膜表面起到隔离猪瓣膜与宿主血液成分接触的作用。同时应用免疫组化的方法验证人细胞外基质覆盖瓣膜的效果。 设计、时间及地点：观察性实验，于2008-08/2009-04在解放军第二军医大学长海医院胸心外科实验室完成。 材料：新鲜猪心脏瓣膜取自上海五丰上食肉联厂，采用酶消化法将猪主动脉瓣叶行脱细胞处理。密度梯度离心法从健康志愿者骨髓中获取骨髓基质干细胞。 方法：用人细胞外基质包被猪去细胞瓣膜支架来构建组织工程瓣膜，再应用前期制备的抗人细胞外基质单克隆抗体，采用免疫组织化学染色鉴定人细胞外基质在体外构建的组织工程瓣膜上的黏附情况。以未构建的去细胞猪瓣叶为对照组。 主要观察指标：①组织工程瓣膜上种子细胞的黏附和生长情况。②免疫组织化学法检测人细胞外基质在瓣膜上覆盖的效果。 结果：光镜下观察瓣膜表面均形成了细胞层。扫描电镜所见细胞排列欠规则，未完全覆盖去细胞瓣叶表面，可见裸露的瓣膜支架。免疫组织化学方法检测构建的瓣膜表面呈阳性反应证明有人的细胞外基质黏附，而对照组呈阴性证明了检测方法具有特异性。 结论：体外构建的组织工程瓣膜上检测到有人的细胞外基质黏附。 关键词：组织工程心脏瓣膜；细胞外基质；黏附

Tissue engineered heart valve construction by coating human extracellular matrix onto decellularized porcine heart valve scaffold

BACKGROUND: Immunological rejection is caused following implantation of decellularized porcine heart valve. The mechanism is highly related to immune activation induced by the contact between extracellular matrix and host. OBJECTIVE: To isolate decellularized porcine heart valve from host blood using self-extracellular matrix and to identify the effect of human extracellular matrix using immunohistochemistry. DESIGN, TIME AND SETTING: An observational study was performed at the Laboratory of Cardiothoracic Surgery, Changhai Hospital, the Second Military Medical University of Chinese PLA from August 2008 to April 2009. MATERIALS: The fresh porcine heart valves were obtained from Shanghai Wufengshangshi Slaughter House; decellularized porcine aortic valves were treated with enzymatic procedure; bone marrow mononuclear cells were harvested by Ficoll density gradient centrifugation. METHODS: The tissue engineering heart valve was constructed by decellularized porcine heart valve scaffold coated with human extracellular matrix. The adhesion of human extracellular matrix to tissue engineering heart valve in vitro was identified by anti-human extracellular matrix monoclonal antibody using immunohistochemistry. Non-constructed decellularized porcine heart valve was considered as the control. MAIN OUTCOME MEASURES: Growth and adhesion of seed cells; the effect of human extracellular matrix adhered on porcine valve scaffold. RESULTS: Intercellular layer was observed on the surface of heart valve under optic microscope. Scanning electron microscope indicated that cells arranged in disorder and partially covered on the heart valve, so naked heart valve scaffold was still observed. Immunohistochemistry demonstrated that positive expression showed on the surface of heart valve, suggesting that the human extracellular matrix was adherent; however, the negative expression was detected in the control group, suggesting the specificity of detecting method. CONCLUSION: The adhesion of human extracellular matrix is defected on tissue engineering heart valve.