脂肪间充质干细胞分化为内皮细胞并体外构建组织工程心脏瓣膜

Adipose derived mesenchymal stem cells differentiate into endothelial cells and construction of tissue-engineered heart valves in vitro

背景:组织工程心脏瓣膜是利用组织工程技术将种子细胞种植于瓣膜支架上所构建的一种人工瓣膜,目前国内外研究主要集中于种子细胞来源及支架选择上。目的:探讨人脂肪间充质干细胞体外向内皮细胞诱导分化后的细胞作为种子细胞,脱细胞猪主动脉瓣膜作为支架体外构建组织工程心脏瓣膜的可行性。方法:利用吸脂术采集脂肪组织,分离、培养脂肪间充质干细胞,流式细胞仪鉴定细胞表型;免疫细胞化学方法及RT-PCR检测细胞分化标志物;应用TritonX-100联合胰蛋白酶的方法制备脱细胞猪主动脉瓣支架,将体外培养扩增的诱导分化后的内皮细胞种植于支架上构建组织工程心脏瓣膜,光镜及电镜下观察组织工程心脏瓣膜的组织学结构。结果与结论:脂肪组织分离培养的脂肪间充质干细胞向内皮细胞诱导分化后表达CD31、CD34、CD144、Ⅷ因子和内皮型一氧化氮合成酶等内皮细胞特异性抗原;脱细胞猪主动脉瓣膜支架脱细胞完全,弹力纤维及胶原纤维保持完整;构建的组织工程心脏瓣膜可见支架上排列连续的单细胞层。提示脂肪间充质干细胞在体外向内皮细胞诱导分化后已初步具有内皮细胞功能,在脱细胞猪主动脉瓣膜支架上生长良好,可以在体外初步构建组织工程心脏瓣膜。

BACKGROUND： Tissue-engineered heart valves are the hotspot of artificial heart valve replacement studies. It utilizes tissue-engineering technology which can construct artificial valves by planting seed cells in valve scaffolds. The current domestic and foreign studies mainly focus on the selection of seed cells and valve scaffolds. OBJECTIVE： To explore the feasibility of constructing tissue-engineered heart valves in vitro by seeding endothelial cells differentiated from adipose derived mesenchymal stem cells in decellularized porcine aortic valve scaffolds. METHODS： The human adipose tissue was collected by liposuction. Then the adipose derived mesenchyrnal stem cells were isolated and cultured, and the cell phenotype was identified by flow cytometry. The cell differentiation markers were detected by immunocytochemistry and RT-PCR. The decellularized porcine aortic valve scaffolds were made by Triton X-100 combined with trypsin digestion procedure. Tissue-engineered heart valves were constructed by seeding the endothelial cells differentiated from adipose derived mesenchymal stem cells on the decellularized porcine aortic valve scaffolds. Morphological characteristics of tissue-engineered heart valves were observed under light and electron microscopes. RESULTS AND CONCLUTION： The endothelial cells differentiated from adipose derived mesenchymal stem cells could express endothelial markers, such as CD31, CD34, CD144 and VIII factor and endothelial nitric oxide synthetase （eNOS） Porcine cells could no longer be detected on decellularized porcine aortic valve scaffolds with elastic fibers and collagenous fibers were well preserved. Endothelial cells formed a continuous monolayer on the surface of the tissue-engineered heart valves. Results indicate that the endothelial cells differentiated from adipose derived mesenchymal stem cells have some endothelial function and can proliferate well on the decellularized porcine aortic valve scaffolds. It is feasible to construct tissue-engineered heart valves in vitro by planting the endothelial cells differentiated from adipose derived mesenchymal stem cells on the decellularized porcine aortic valve scaffolds.