摘要
背景:成年哺乳动物脊髓损伤(Spinal cord injury, SCI)后的修复十分困难,我们曾应用多种神经组织移植修复成鼠脊髓损伤,效果均不理想。
神经干细胞(Neural stem cells, NSCs)因其分布的广泛性及多分化潜能,被视为中枢神经系统替代治疗的理想材料。但单纯NSCs移植后很快弥散流失或形成细胞团,出现营养缺乏和代谢障碍,不能形成功能性的修复。利用组织工程技术构建神经干细胞/支架复合物可以克服这些缺点,为细胞附着提供锚靠点,以利于其生长、分化和代谢。但急性SCI的微环境会抑制NSCs向神经元分化,甚至对其存活都会有负面影响,NSCs主要分化成星形胶质细胞而非神经元,神经干细胞/支架复合物作为修复材料的潜能受到巨大挑战。如何改善局部环境,如何诱导神经干细胞更多地向神经元表型分化,是脊髓组织工程研究中亟待解决的问题。
处在发育期的胚胎脊髓(Fetal spinal cord, FSC)允许自身神经干细胞大量增殖及向神经元表型分化。同时,FSC中存在高浓度的NT-3和BDNF等神经营养因子,具有诱导神经干细胞向神经元方向分化的能力,并可提供其他有益的蛋白质成分、神经化学因子或细胞外基质,起到调控和协同作用,使脊髓固有的各种细胞成份最终达到合适的比例。
鉴于上述,本实验将NSCs接种在PGA支架上,再提供胚胎脊髓提取液(Fetal spinal cord extracts, FE)模拟NSCs发育的胚胎性微环境,提供生长分化所需的神经化学因子或细胞外基质,促进向神经元表型分化,体外构建了一种包含神经元和胶质细胞等成份、具有一定组织形态和功能的复合神经组织,我们暂将其命名为组织工程化脊髓(Tissue-engineered spinal cord, TSC)。如此,修复急性SCI时通过移植这种在体外己完成分化形成神经元、星形胶质细胞和少突胶质细胞的组织,就有可能避免体内的复杂环境对干细胞分化的不确定性影响,提高修复成年鼠脊髓损伤的效果。
目的:利用组织工程技术,研究和开发能修复成年鼠损伤脊髓的组织工程材料,为脊髓损伤的治疗提供新思路、新方法。
方法:1.胚胎脊髓神经干细胞的分离培养与鉴定。分离和克隆扩增孕14天SD大
Background: The repairing of spinal cord injury (SCI) has always been a hot spot in medical research. We have used multiple nervous tissuse to repair SCI, but less success has been achieved in functional repair of the injured adult spinal cord.
Recently, much has been learned about the biology of neural stem cells (NSCs). NSCs isolated from the brain and spinal cord of both neonatal and adult rats reportedly retain the potential self-renewing and to differentiate into neurons, astrocytes, and oligodendrocytes. It is a kind of ideal materials to substitute lost central neural tissues. Interestingly, cell implantation alone failed to generate functional tissue, this may be due to cells lacking a three-dimensional substrate disperse rapidly and are not uniformly distributed across the surgically created gap. The use of a threedimensional scaffold may provide anchorage sites for cell attachment and cues for differentiation. However, the microenvironment of acute spinal cord injury may inhibit the appearance of neurons, mainly the NSCs differentiated into glial cells, few into neurons. The potency of NSCs-scaffold complex as a reparing materials to be challenged. In the study of spinal cord tissue engineering, there are lots of urgently problem, such as how to improve local environment, and how to induce NSCs differentiate into neurons.
In the developing fetal spinal cord (FSC), endogenous NSCs multiply rapidly and differentiated into neurons phaenotype. Meanwhile, FSC could excreting high-concentration neurotrophic factor, such as BDNF, NT-3, and so on, can facilitate NSCs to differentiate into more neurons. FSC also can provid helpful proten composition, neurochemistry factors or extracellular matrix, to regulate propriospinal cellour reconstituent to a suitable proportion.
Based on the reasons above, we implanted NSCs into polyglycolic acid (PGA) scaffold, subsequently provid fetal spinal cord extracts (FE) to induce cell differentiation, constructed a kind of nervous tissue complex, contain neurons, astrocytes, and
引文
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