【作者】 孔征东；

【导师】 张志英； 张传森； 杨向群； 李玉泉； 熊绍虎；

【作者基本信息】 第二军医大学 ， 人体解剖与组织胚胎学， 2010， 硕士

【摘要】 研究背景和意义：外周神经损伤是临床常见损伤,外周神经缺损的修复是世界性的难题,主要是神经生长速度慢和效应器、感受器萎缩快的矛盾。如何提高神经再生的速度和质量,寻找新的修复方法是外周神经损伤迫切需要解决的瓶颈问题。目前外周神经缺损的治疗策略,是依据外周神经损伤再生的理论,通过提供一个神经纤维再生通道、营造有利于神经纤维再生微环境等,涉及神经导管及细胞因子的应用、细胞或神经移植、以及近年来兴起的组织工程化外周神经等。这些治疗方法已经在动物实验或临床上取得了可喜的效果,但由于神经生长速度较慢,效应器的退化较为严重,用于修复长距离神经缺损效果不满意。鉴于此,我们提出了“桥接神经元修复外周神经缺损”的设想,即通过神经元接力的方法,加快神经到达效应器、感受器的时间,减少效应器、感受器病变程度,提高神经损伤修复的效果。为验证此设想,本研究从临床实用出发,拟以骨髓源性神经组织定向干细胞(NTCSC)源性神经元细胞为种子细胞、以同种异体脱细胞神经支架为支架材料,构建神经元性组织工程化神经,用于修复犬坐骨神经损伤,探讨神经元性组织工程化神经修复外周神经的机制及效果,旨在为外周神经缺损的修复提供一个新的方法。第一部分NTCSC的培养、诱导分化及鉴定研究目的：研究骨髓源性神经组织定向干细胞(NTCSC)的分离、培养、鉴定及向神经元细胞的诱导分化,探讨作为神经元细胞来源种子细胞的可能性。研究方法：取beagle犬自体骨髓,全骨髓培养,留取贴壁细胞,添加BFGF和EGF的无血清培养液继续培养,待细胞聚集呈球状时,吹打散开后传代培养扩增,细胞鉴定。分别用RA+SHH和NRG1诱导分化,观察诱导细胞形态、生长情况,检测其表面标志。结果：传代细胞悬浮、球形生长、增殖能力旺盛、Nestin阳性,为骨髓源性神经组织定向干细胞。RA+SHH诱导分化后,神经球贴壁、分散、β-Tubulin阳性、NeuN阳性,为神经元样细胞。NRG1诱导后,神经球散开、长出较长的突起、表达S-100,为雪旺氏细胞。结论：NTCSC扩增能力强、有神经系细胞的特性、易于向神经元样细胞以及雪旺氏细胞分化,是较为理想的神经元细胞来源的种子细胞。第二部分神经元性组织工程化神经的的构建。研究目的：研究脱细胞支架制备、组织工程化神经的构建,制备神经元性组织工程化外周神经移植体。研究方法：取Beagle犬坐骨神经6cm,修剪、0.3%SDS脱细胞5天、RNA酶和DNA酶消化2天、蒸馏水反复漂洗5天、密封、60Co辐照灭菌、4℃保存。将诱导的雪旺氏细胞和神经元样细胞按照1：1的比例混合成细胞悬液,用微量注射器种植到支架中,制成神经元性组织工程化神经移植体。结果：大体观察见脱细胞神经支架外形完整,乳白色,H-E染色未见到细胞成分,纤维连续无断裂。组织工程化神经移植体柔软、饱满,种植细胞在支架内生长,迁移、分布均匀。结论：成功构建了神经元性组织工程化外周神经移植体。第三部分神经元性组织工程化神经的体内研究研究目的：研究神经元性组织工程化外周神经移植修复坐骨神经6cm缺损的效果,探讨桥接神经元修复外周神经长段缺损的可行性。研究方法：制备犬双坐骨神经6cm缺损模型6只,神经元性组织工程化神经、雪旺氏细胞性组织工程化神经和自体神经分别移植到相应的犬4侧缺损的坐骨神经上。术后进行动物行为学观察。术后13周,三组犬进行肌电图检测。4侧神经元性组织工程化神经移植组行辣根过氧化物酶逆行追踪；全部动物处死、取坐骨神经、切片、免疫组织化学染色、甲苯胺蓝染色、透射电镜检测；取腓肠肌、检测蛋白质的含量。结果：术后犬下肢不能活动,自术后2周始,三组犬均出现下肢肌肉萎缩、足底溃疡。术后8周,神经元性组织工程化外周神经移植犬对针刺有反应、下肢能触地行走,自体神经移植组术后10周恢复感觉运动功能,雪旺氏细胞性组织工程化神经移植组术后13周恢复感觉运动功能。肌电图检测表明：神经元性组织工程神经恢复效果理想。神经元性组织工程化外周神经移植犬腓肠肌中注射HRP后,神经移植体内可见天青色的团块。免疫组织化学显示,神经元性组织工程化神经移植体中NeuN阳性。甲苯胺蓝染色,神经元性组织工程化神经移植体中髓鞘数量多于雪旺氏细胞性组织工程化神经移植组。透射电镜显示神经元性组织工程化神经移植体中有突触的形成。考马斯亮蓝检测腓肠肌蛋白质含量,实验组>自体神经移植组>雪旺氏细胞移植组。结论：种植的干细胞源性神经元样细胞可以在外周神经微环境内存活、并维持神经元的功能,桥接神经元修复外周神经缺损的效果优于传统组织工程化神经和自体神经移植,桥接神经元修复外周神经缺损可行。更多还原

【Abstract】 Background and Significance:Peripheral nerve injury is common clinical damage, and the repair of peripheral nerve defects is a worldwide problem. Difficulty is Contradictions between the slow growth rate of nerve and the fast atrophy of effectors.The improvement of speed and quality of nerve regeneration is an urgent need to solve the repair of nerve injury. At present the treatment of peripheral nerve defect strategy is based on the theory of regeneration of peripheral nerve injury, by providing a channel, and creating a favorable micro environment for nerve fiber regeneration, and it is involved the application of Neural tube and cytokines, as well as the recent emergence of tissue engineering nerve. These treatments have been achieved gratifying results in animal experiments and clinical trials. But for a long nerve defect. But this recovery rate was not yet satisfied. Effectors of the degeneration is still very severe. In view of this, we have proposed a "neurons bridging to repair of peripheral nerve defects" the idea is that by neurons relaying, the time for the axons arriving to the effctors is reduced and the regenerations of effecors is slow down.To test this idea, this study started from the clinical utility, intended to construct neuronal tissue engineering nerve with bone marrow-derived neural tissue committed stem cells (NTCSC)-derived neuronal cells and allogenic acellular nerve, and repair the dog sciatic nerve injury. mechanisms and effectsof repairing peripheral nerve with neuronal tissue engineered nerve, aiming to provide a new approachof repairing peripheral nerve defects.Section 1 culture, induced differentiation and identification of bone marrow-derived NTCSC.Objective:To study separation, culture, identification of bone marrow-derived neural tissue committed stem cells (NTCSC), as well as induction and differentiation to neurons, the separation, culture, identification, and the induction and differentiation of neurons, and to discuss the possibility of neuronal cells as a source of seed cellsMethods:The cells are Harvested from dogs bone marrow, and first cultured in a serum culture medium for 7days. Later the cells were moved to a serum-free medium cantaining with BFGF and EGF. After the cells grow to form cells sphere. Disperse the cells sphere and continue to cuture the cells sphere. And this cells sphere is bone marrow-derived NTCSC. Let adherent neurosphere and use RA+SHH to induce the NTCSC to neuronal differentiation, as well as NRG1 to induce to Schwann cell differentiation. At different time observe cell morphology, growth, detect surface marker of cells.Results:The bone marrow-derived neural tissue committed stem cells (NTCSC) suspend, and grow spherically and proliferative capacity of cells is strong. NTCSCs express CXCR4-positive, Nestin-positive. After RA+SHH induction, the neurospheres adherent and expressβ-Tubulin-positive and NeuN-positive. With NRG1-induced, the neurospheres spread out, grow long And express S-100 positive.Conclusion:NTCSC has great expansion capability and have characteristics of neuralline, NTCSC can differentiate into neurons and Schwann cells, as a result NTCSCs is an ideal seed cells for tissue engineering nerve.Section 2 Construction of neuronal tissue engineering.Objective:Prepare a safe nerve scaffold of non-immune response, suitable bio-mechanical properties. Seed cells to nerve scaffold and Prepare tissue-engineered nerve. Transplant the nerve to the beagle’s leg.Methods:6cm sciatic nerve were havested from died beagle dogs.The nerve were Rinsed with 0.3% SDS for 5 days and RNA enzymes, DNA enzyme for 2 days, and rinsed with distilled water Repeatedly for 5 days. The Acellular nerve graft were sterilizated by 60Co irradiation, and then conserved at 4℃. Induced Schwann cells and neuron-like cells were mixed into the cell suspension in accordance with the ratio of 1:1, and injected into the scaffold with micro-syringe. And now the neuronal tissue-engineered nerve is prepared.Results:acellular nerve scaffold is of complete shape, and did not contain cells. The Fibers of scaffold were continuous and of non-fracture. After the cultivation of cells, tissue engineering nerveare soft and plump. The nerves were well suture with the scatic nerves. The planted cells grew in the scaffold and migration, distribution was observed.Conclusion:The biomechanical properties of Acellular nerve graft is good, containing less cellular components. The Acellular nerve graft is a safe carrier for nerve tissue engineering.Section 3 the in vivo research of neuronal tissue-engineered nerve.Objective:To study the effect of repairing the scatic nerve 6cm defects with neuronal tissue engineering nerve and disscuss the feasibility of repairing the long segment of peripheral nerve defects with Bridging the neurons.Methods:Prepare 6 canine models of 6cm defects of two Hind legs scatic nerve. Neuronal tissue engineering nerve、schwann cell tissue engineering nerve and autologous nerve graft were transplanted into the 4 scatic nerve defects. Animal behavior were observed after the operations. After 13 weeks, EMG was examined. 4 sides neuronal tissue engineered nerve graft group underwent retrograde horseradish peroxidase. after killing all the animals, immunohistochemical stain, toluidine blue staining, transmission electron microscopy detection and protein content detection were taken.Results:The left lower limb of all three groups dogs can not move after operations. After 2 weeks, muscle atrophy were found in the limbs of three groups begin muscle atrophy. After 8 weeks the experimental group dogs began to react to acupuncture, and lower limbs can touch the ground walking. Sensorimotor all recovered. Autograft control group recovered after 10 weeks and Schwann cell transplantation group were recovered after 13 weeks.After HRP were injected Gastrocnemius muscle of neuronal tissue engineering Nerve Graft dog, blue clumps is visible in nerve graft. Immunohistochemistry showed that neuronal tissue engineered nerve graft in NeuN positive.Toluidine blue staining showed that the number of myelin sheath in neuronal tissue engineered nerve graft is more than Schwann cells in tissue engineering nerve group.Transmission electron microscopy revealed synapse formation in neuronal tissue engineered nerve graft. Coomassie blue detection of protein content showed that protein content of gastrocnemius muscle in the experimental group> nerve autograft group> Schwann cell transplantation group.Conclusion:Transplanted stem cell-derived neuron-like cells can survive in the peripheral nerve microenvironment and maintain neuronal function. Repairing peripheral nerve defects by bridging the neurons is better than the traditional neural tissue engineering and autologous nerve graft. And this method is feasible.更多还原