复合细胞膜片构建血管化组织工程三维全喉支架的实验研究

The construction of tissue engineered larynx is an effective way to solve the problem after total laryngectomy. But how to vascularize the tissue engineered larynx is still a tough problem. Most of the methods to produce vascularized tissue revolve around the selection or development of physical and biological materials, cells and agents. Our previous study have constructed a wholly decellerized laryngeal scaffold which has low immunogenicity, better biomechanical structure, and could promote cell regeneration. The scaffold could be an appropriate laryngeal scaffold for vascularization. It is shown that the coculture of the endothelial cells with mesenchymal stem cells produces a longlasting vascular network. The adipose-derived stem cell is one of the mesenchymal stem cell, which possess vascular properties and can give rise to both endothelial and perivascular cells. However, it is not clear if the coclutured adipose-derived stem cell and endothelial cells with the decellularized laryngeal scaffold could construct a vascularized tissue engineered larynx. So in this future research, the project intends to use the cell sheet technology construct endothelial cell sheet and adipose-derived stem cell sheet, and observe the composite endothelial cell sheet and adipose-derived stem cell sheet promote revascularization of the decellerized laryngeal scaffold in vitro and in vivo by cell culture, immunohistochemistry, RT-PCR and other methods. It will provide theoretical basis and experimental evidence for constructing vascurized tissue engineered organs.

构建组织工程全喉是解决全喉切除后疾患的有效途径，组织工程全喉的血管化是组织工程全喉应用于临床的重点和难点。构建血管化组织工程器官需要合适的生物物理材料，细胞和促进细胞生长的细胞因子。我们前期构建的去细胞全喉软骨支架具有较低的免疫原性，较好的生物力学结构，同时有利于细胞再生，可为构建血管化的组织工程全喉提供生物支架。近期国内外研究发现内皮细胞与间质干细胞共培养可在体内形成稳定的血管网，而脂肪源性干细胞作为间质干细胞的一种，同时具有血管生成的特性，能促进内皮细胞和血管周细胞生长。然而脂肪源性干细胞和内皮细胞能否与去细胞全喉支架复合构建出血管化的组织工程全喉有待进一步证实。为此，本项目拟采用细胞膜片技术，通过细胞培养，免疫组织化学，RT-PCR等方法，观察内皮细胞膜片和脂肪源性干细胞膜片在体外和体内促进去细胞全喉支架再血管化的作用，为组织工程器官的血管化提供理论基础和实验依据。