基于MEMS工艺构建生物人工肾小球的基础研究

The function and the structure of glomeruli have been clearly demonstrated.The glomerular filtration membrane, which is comprised of vascular endothelial cells, glomerular basal membrane and podocytes, plays a major role in the selective filtration of kidney. With the development of tissue engineering and the application for nanotechnology and bioMEMS technology, the construction of bioartificial kidney glomerulus becomes possible. In our previous research, with the use of advanced technology such as sacrificial layer technology, anisotropic etching and film deposition, a model of composite nanoporous filtration membrane ，which being coated collagen, was intented to simulate glomerular basal membrane. It has indeed shown great biocompatibility and similar filtration function to normal kidney basal membrane. Here, to further construct a better composite model of nanoporous filtration membrane which can satisfy the physiological characteristics of glomerular filtration membrane we improved our processing technics through cultivating endothelial cells and podocytes in both sides of composite nanoporous filtration membrane and then assembling them into miniature bioreactor. The filtration of water and different kinds of solute in this new bioartificial kidney glomerulus will been validated by rat kidey failure models. The creation of bioartificial kidney glomerulus will provide an ideal model for further researching kidney physiology and develpoing new medicine, pave the way for the development of micromation and implantation of artficial filter and eventually make the artificial kidney possible.

肾小球的结构和功能已基本明晰，发挥选择性通透作用的主要是血管内皮细胞、基底膜和足突上皮细胞构成的滤过膜。随着组织工程学的迅猛发展和生物微电子机械系统（BioMEMS）纳米技术的应用，使得模拟人工生物肾小球成为可能。我们在前期的工作中采用牺层技术、各向异性刻蚀、薄膜图形化沉积等方法初步构制了复合结构多孔过滤膜，包被胶原蛋白，人工仿制基底膜，显示了良好的生物相容性和接近生理的滤过效应。在本研究中将进一步优化加工工艺，构建最符合肾小球滤过膜生理特性的复合结构多孔过滤膜，并分别将血管内皮细胞和足细胞培养在复合结构多孔过滤膜上下两面，形成人工制作的肾小球滤过膜，再将其组装到微型生物反应器中合成生物人工肾小球，利用大鼠肾衰模型验证生物人工肾小球对水和各类溶质的滤过效应。生物人工肾小球的构建，将为深入研究肾脏生理和研发新药提供理想的体外模型，为微型化及植入式人工滤器的研发乃至最终合成生物人工肾提供可能。

肾小球的结构和功能已基本明晰，发挥选择性通透作用的主要是内皮细胞、基底膜和足突上皮细胞构成的滤过膜。随着组织工程学的迅猛发展和生物微电子机械系统（BioMEMS）纳米技术的应用，使得模拟人工生物肾小球成为可能。我们采用牺层技术、各向异性刻蚀、薄膜图形化沉积等方法初步构制了复合结构多孔过滤膜。并用胶原蛋白进行包被，前期研究显示了良好的生物相容性。在本研究中我们优化了加工工艺，进一步缩小了滤过孔直径，并分别将内皮细胞和足细胞培养在复合结构多孔过滤膜上下两面，形成人工制作的肾小球滤过膜。并利用扫描电镜和免疫荧光染色技术对其细胞生长情况进行直接观察。之后，将人工制作的肾小球滤过膜组装到微型生物反应器中合成生物人工肾小球。并探究测量了其在不同的跨膜压下，对于免疫球蛋白IgG（160kd），白蛋白（68kd），维生素B12（1.36kd）的滤过特性。在低跨膜压（0.05 PSI）的情况下，球蛋白IgG的表观清除率为0.011，白蛋白的表观清除率为0.106，维生素B12为1。当提高跨膜压后，其表观清除率也会逐渐升高。生物人工肾小球的构建，将为深入研究肾脏生理和研发新药提供理想的体外模型，为微型化及植入式人工滤器的研发乃至最终合成生物人工肾提供可能。