【作者】 李肖；

【导师】 何际平； 徐琦；

【作者基本信息】 华中科技大学 ， 控制科学与工程， 2017， 博士

【摘要】 由于意外事故、自然灾害、恶性疾病,导致我国每年新增大量外周神经损伤患者。如果未能及时进行有效的神经修复和康复治疗,将影响患者运动和感觉功能,甚至导致其生活和工作能力丧失。外周神经损伤的治疗分为两种,一种是肢体完整,可通过神经修复,使断开的神经末端重新连接在一起,恢复自然的神经功能;另一种是已经截肢,需安装智能假肢,可通过神经接口技术实现人体与假肢信息互联,重建感觉运动功能。将神经修复或神经接口技术与组织工程技术结合,用于医治神经损伤表现出极大的应用前景。但是,无论是长距离的神经修复,还是新型的神经接口,都需要大量的自体神经组织。为了解决自体神经组织来源不足、大小不匹配等问题,我们开发一种新的神经培育装置,具体做了以下工作:研制了一种神经牵拉培育装置,用于对形成突触连接的神经细胞进行牵拉培养。该装置由控制系统和机械系统两部分组成,其设计原理是:先将神经细胞分别培养在牵拉膜与底膜上;当细胞之间形成突触连接后,通过控制器控制步进电机转动,通过一系列机械传导带动牵拉膜的运动,进而对附着在牵拉膜上的神经轴突进行牵拉;根据需要,经过一段时间的牵拉培养,即可得到一段长度合适、排列规则的神经组织。为了保证神经牵拉生长过程的稳定性,实验前我们对牵拉装置进行了位移测试。通过对0.5微米、1微米和2微米的步距进行统计学分析,最后发现1微米的步距适合神经细胞的牵拉培养。神经牵拉培育装置的底膜与牵拉膜采用聚三氟氯乙烯薄膜,该材料生物兼容、透明、不易发生形变,满足轴突牵拉的需求,但该薄膜须进行表面修饰后才能用于神经细胞的培养。本文分别用左旋多聚赖氨酸(Poly-L-Lysine,PLL)、右旋多聚赖氨酸(Poly-D-Lysine,PDL)、鼠尾胶、层粘蛋白等修饰材料进行分组对比分析,针对背根神经节(Dorsal Root Ganglion,DRG)的离体培养过程,通过统计各组贴壁率、生长速度和单位长度末梢数,结果发现PDL组、PDL+层粘蛋白组和PDL+鼠尾胶组适合后期的神经牵拉培养实验。随后,我们在神经牵拉培育装置内对大鼠背根神经节进行了静态培养与牵拉培养,在较短时间内成功培育出长度合适、排列规律的神经束。通过荧光染色与形态学观察,确定了所培育的神经成份是轴突,且细胞骨架结构完整、没有断裂,具备传导神经信号的物质基础。此外,我们还在神经牵拉培育装置内进行了大鼠背根神经节的三维静态培养与三维牵拉培养,结果表明该装置也适合神经细胞的三维培养,且鼠尾胶形变对轴突生长方向有一定导向作用,从而为牵拉神经的封装和神经三维牵拉培养提供了思路。鉴于目前尚无电生理仪器可用于直接检测牵拉生长条件下轴突的电信号,为研究牵拉生长后神经细胞的神经信号传导能力与放电特性,我们建立了牵拉生长的无髓鞘背根神经节细胞仿真模型,比较了静态培养与牵拉培养条件下神经细胞各组分神经信号的传播速度、幅值,分析了牵拉培养的神经元对不同刺激频率和刺激幅值的敏感性,以及T型分支对神经信号传播的影响。该仿真结果可为将来的电生理实验提供一定的理论指导。更多还原

【Abstract】 Because of rising traumatic accidents and diseases,the number of patients suffer from peripheral nerve injury is increasing.If they fail to carry out effective neural repair and rehabilitation therapy,it will affect the patient’s motor and sensory functions,and bring much trouble to their life.There are two kinds of treatments for peripheral nerve injury.If the patient’s limbs are intact,the surgical procedure can be performed to suture the disconnected nerves to restore the natural neural function.As for amputees,neural interface technology can be used to splice nerves and electrical wires together in a way that allows them to control an artificial limb as if it was a natural extension of the body.Neural repair and neural interface technology based on tissue engineering have shown promising prospects in the treatment of nerve injury.However,both of the two treatments need autologous nerve to stimulate axonal regeneration and extension into target tissues,which are limited by the supply of donor nerves.In the dissertation,the main contributions were as follows:We had designed and fabricated a new miniaturized bioreactor system for stretch growth of integrated axon tracts.The device was composed of two parts:the control system and mechanical system.Neurons were firstly placed on two adjoining substrates and formed new synaptic connections between each other.Then the axon bundles across the border between the top and bottom membranes were stretched in a stepwise fashion by a microstepper motor system.After several days’ stretch,the axon tracts could reach specific length that can be used to create living nervous tissue constructs.By measuring the linear displacement with 0.5 μm,1 μm and 2 μm step,we found the axon stretch system taking 1μm every step was the most appropriate.The substrate of axon expansion chamber was made of Aclar film,which was a transparent film,suitable for axon stretch growth after surface modification.In order to get more appropriate neuronal culture and stretch conditions,the Aclar films were respectively treated with PLL,PDL,laminin,type 1 rat-tail collagen,both PDL and lamini,both PDL and collagen.Through the statistical analysis of the DRGs cultured in the different groups,we found the elongator substrates coated by PDL,both PDL and rat-tail collagen,or both PDL and laminin could maximize the number of axons bundles.And they were the best methods to modify the Aclar film for axon stretch growth.The DRGs were cultured in the bioreactor,and the bridging axons adapted to the stretch by increasing their length from 500 μm to become about 6 mm long over 7 days of stretch growth.Immunocytochemical analysis confirmed that major cytoskeletal constituents were present along axons.Then we cultured DRGs in a 3 dimensional(3D)fashion and found the axons also grew very well in the bioreactor,which provided some new thoughts for the encapsulation of stretch growth nervous tissue.There is no direct electrophysiological examination devices for axons under extreme stretch growth conditions.In order to investigate the ability of DRG neurons to generate and convey action potentials after a period of rapid axon stretch growth in vitro,and what stimulating parameters can evoke action potential,we have used computational tools to simulate the stretch growth neurons.In the simulation,we had analyzed the spike amplitude and conduction velocity in both static culture neuron and stretch growth neuron.And we also analysed the sensitivity of spike propagation in the stretch growth neuron to different stimulating frequencies and stimulating voltages.Finally,we had investigated how t-junction influenced the spike propagation in neurons.It could provide some guidelines for the electrophysiological experiments in future.更多还原