Noggin基因调控重编程蛋白诱导性多能干细胞向神经细胞分化移植修复SCI的实验研究

Spinal cord injury (SCI) is a common disease with high disability rate in clinic. The current treatment of SCI is not ideal, and it is one of the most difficult problems to be solved in medical field. Recent studies show that recuperating missing neurons and oligodendrocytes is the key element of SCI repair. This project aims to obtain two reprogramming proteins of Oct4 and Sox2 gene expression product with the gene engineering technology, and utilize them to induce rat dermal fibroblasts into proteins-induced pluripotent stem cells (p-iPSCs). Noggin gene plays an important part in the neural differentiation process, so p-iPSCs will be modified and induced by Noggin to differentiate into neurons and oligodendrocytes. Then Noggin-modified p-iPSCs will be implanted into SCI rats to repair SCI through migrating and homing to replace missing neurons and oligodendrocytes via vein graft and local injection. Through obseving the ingegration of neurons and oligoendrocytes derived from Noggin-modified p-iPSCs with the normal nerve tissue and the effect of structure regeneration and functional recovery of SCI, we will explore the potential and mechanism of p-iPSCs differentiating into neurons and oligodendrocytes, thus provide optimal cell source for the cellular transplantation treatment of SCI.

脊髓损伤临床上常见,现行的治疗方法疗效不理想,致残率高,是医学领域亟待解决的难题。现有研究显示影响SCI修复的关键要素是神经元和少突胶质细胞缺失。本项目拟借助基因工程技术在获取Oct4和Sox2两个关键性多能相关基因表达产物重编程蛋白的基础上,直接应用重编程蛋白诱导大鼠皮肤成纤维细胞产生p-iPSCs,将具有独特神经分化诱导功能的Noggin基因导入修饰p-iPSCs，调控该细胞向神经元和少突胶质细胞分化,并将Noggin基因修饰的p-iPSCs采用静脉移植和局部植入方式移植修复SCI，通过迁移、归巢替代SCI所致的神经元和少突胶质细胞缺失,通过观察与SCI邻近正常神经组织的整合状况、对受损脊髓结构重构及功能恢复的影响，探讨p-iPSCs向神经细胞的分化潜能、分化机制及移植p-iPSCs修复SCI的价值，从而为细胞移植修复SCI提供理想的细胞来源，使SCI的治疗有所突破。

脊髓损伤临床上常见,现行的治疗方法疗效不理想,致残率高,是医学领域亟待解决的难题。现有研究显示影响SCI修复的关键要素是神经元和少突胶质细胞缺失。本项目拟直接应用重编程蛋白诱导大鼠皮肤成纤维细胞产生p-iPSCs,将具有独特神经分化诱导功能的Noggin基因导入修饰p-iPSCs，调控该细胞向神经元和少突胶质细胞分化,移植修复SCI。在实验中，为了解决p-iPSCs的重编程效率低，且在培养过程中易分化，影响种子细胞的获取和后续的实验的问题，我们尝试通过借助microRNAs cocktail诱导细胞转分化为神经元，结果显示microRNAs cocktails可使成纤维细胞表达map2、nf200、βⅢtubulin等神经元特异性标记物，通过细胞膜片钳技术检测到转分化细胞的钠、钙电流，从而证实通过转分化获得一种类神经元。该转分化方法获得的转分化细胞的效率高达8%。该直接转分化方法避开了ips细胞的多能状态，实现一种无外源基因整合、高效、安全地获取神经细胞的方法，为临床应用奠定一定基础。