HIF-1α基因修饰的MDSCs促进组织工程化海绵体快速血管化及机制的研究

[Abstract]: The special anatomical structure and non-renewable characteristic of cavernous tissue are always the main obstacle to the penile reconstruction. The study of tissue-engineered corpus cavernosum is currently the only way to solve this problem. The existing seed cells and scaffolds are all difficult to constructe cavernous tissue with rich vascular and good physiological functions. In our preliminary experiments, we prove the feasibility which using muscle-derived stem cell (MDSCs) as the seed cell and acellular cavernous matrix as a scaffold to construct the tissue-engineered corpus cavernosum. The key factors are to reduce the antigenicity of the scaffold, promote the activity of the seed cell and the rapid vascularization of the scaffold. On the basis of the preliminary study, we will optimize the scaffold, construct the seed cell lines of lentiviral vector-mediated HIF-1α gene-modified MDSCs, detect the growth, migration, adhesion and proliferation of seed cell and the expression of gene, repair the defect of the corpus cavernous, use MRI, ICP, Immunohistochemistry et al to evaluate the survival of scaffold, angiogenisis, therapeutic effect and explore the molecular mechanisms. This project is expected to build a organized and vascularized “living” scaffold and overcome the difficulties to reconstruct the corpus cavernosum.

阴茎海绵体特殊的解剖结构及无法再生的特性是阴茎重建手术的难点。脱细胞海绵体基质是组织工程化海绵体理想的支架材料。我们前期的研究显示了MDSCs作为种子细胞同脱细胞海绵体基质相结合构建血管化组织化海绵体的可行性，但内部易出现坏死、液化及纤维化等。如何优化支架并促进其快速血管化是提高支架存活的关键。HIF-1α基因是激活新生血管网形成的关键因子。本项目拟采用基因点突变及慢病毒转染技术构建MDSCs/HIF-1α种子细胞系；利用激光打孔技术制备激光微孔脱细胞海绵体基质；利用生物芯片技术、体外三维血管构建技术、蛋白生化技术、病理检测技术、分子影像学技术及电生理技术等，研究种子细胞在低氧环境中增殖分化及旁分泌的情况，探索其促进激光微孔脱细胞海绵体基质快速血管化和体内多向分化的分子机制。本课题有望为构建大块血管化组织化“活”支架提供坚实的实验依据。

阴茎海绵体特殊的解剖结构及无法再生的特性是阴茎重建手术的难点。脱细胞海绵体基质是组织工程化海绵体理想的支架材料。我们前期的研究显示了MDSCs作为种子细胞同脱细胞海绵体基质相结合构建血管化组织化海绵体的可行性，但内部易出现坏死、液化及纤维化等。如何优化支架并促进其快速血管化是提高支架存活的关键。本研究通过构建HIF-1α基因转染的MDSCs，以HIF-1α基因作为激活新生血管网形成的关键因子，促进下游各类血管因子的表达，促进快速微循环的建立。本项目以慢病毒质粒为载体成功构建MDSCs/HIF-1α种子细胞系。在常氧和低氧条件下的对构建的种子细胞生长，增殖进行检测，提示种子细胞组在低氧条件下要优于其他对照组。我们对HIF-1α启动的下游血管因子VEGF，Ang-1的表达检测，提示种子细胞组稳定表达HIF-1α可以启动下游血管因子的上调。本研究构建新的支架材料，即利用3D打印技术制备水溶胶海绵体支架。我们评估对照种子细胞在脱细胞基质支架和3D材料的种植及生长情况。完成MDSCs/HIF-1α作为种子细胞联合3D水溶胶材料裸鼠皮下的动物实验及标本的各类基因及蛋白的检测。本研究旨在检测种子细胞其促进材料支架快速血管化和干细胞在体内多向分化的分子机制。本课题有望为构建大块血管化组织化“活”支架提供坚实的实验依据。