基因工程化神经干细胞结合组织工程材料治疗周围神经损伤的实验研究

发布时间：2018-05-25 14:21

本文选题：基因工程 + 组织工程　；参考：《第三军医大学》2005年博士论文

【摘要】：神经损伤的再生修复一直是神经科学研究的难点和焦点。近年来,随着交通事故和创伤的逐年上升,由此引起的神经损伤,尤其是外周运动神经损伤日益增多,其特点为致残率高,危害性大。由于缺乏治疗神经损伤的理想药物,临床上仅能将断裂的神经吻合,其结构及功能的恢复很不理想。如何有效的治疗外周神经损伤,特别是较长距离的神经损伤,使之有效的实现功能恢复,这也是当前神经科学研究中亟待解决的问题之一。目前的研究表明,周围神经损伤后虽有一定的自我再生能力,但良好的再生修复更需要神经营养因子(neurotrophic factors,NTFs)、基质和神经细胞这三方面因素的协同作用来实现。胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)对多巴胺神经元、运动神经元等多种神经元均具有促进存活和损伤保护的作用,极有希望用来治疗神经损伤和神经系统退行性病变。实施基因治疗将GDNF植入损伤局部,使之持续表达分泌GDNF,就解决了外源性给药作用时间短等的不足。基因治疗要选择适宜的载体细胞。神经干细胞(neural stem cells,NSCs)能在体外大量增殖,并可诱导分化为神经系统的主要细胞——神经元和胶质细胞。因此,NSCs成为基因治疗理想的载体细胞。同时组织工程材料壳聚糖与组织有较好的相容性,能被组织吸收,更能抑制成纤维细胞的生长,防止瘢痕形成,因此它也是应用于神经损伤治疗中的理想材料。本实验运用细胞培养技术、基因转染技术、流式细胞计数、免疫组织(细胞)化学技术、HRP逆行示踪、坐骨神经功能指数,膜片钳电生理技术等方法,分离、培养大鼠大脑皮层NSCs,观察NSCs与壳聚糖、雪旺细胞(Schwann's cells,SCs)共培养条件下生长、分化的情况。并将GDNF基因在体外转染到NSCs中,筛选、扩增后移植到经过壳聚糖管套接的大鼠坐骨神经离断模型中,以满足神经再生修复的三大因素,观察评价治疗效果,为损伤神经的治疗探索新的可行性。主要结果如下: 1.本实验分离、培养胚胎大鼠大脑皮层NSCs。细胞呈球状悬浮生长,细胞克隆
[Abstract]:The regeneration and repair of nerve injury has always been the difficulty and focus of neuroscience research. In recent years, with the increasing of traffic accidents and injuries, the nerve injury, especially the peripheral motor nerve injury, is increasing day by day, which is characterized by high disability rate and great harm. Due to the lack of ideal drugs for the treatment of nerve injury, the broken nerve can only be anastomosed clinically, and the recovery of its structure and function is not satisfactory. How to effectively treat peripheral nerve injury, especially the long distance nerve injury, so that it can effectively achieve functional recovery, which is one of the problems to be solved in the current neuroscience research. Current studies have shown that although the peripheral nerve injury has a certain ability of self-regeneration, good regeneration and repair need the cooperation of neurotrophic factor neurotrophic factors NTFsI, matrix and nerve cells. Glial cell line-derived neurotrophic factor (GDF) can promote the survival and protection of many kinds of neurons, such as dopamine neurons, motor neurons and so on. When gene therapy was carried out, GDNF was implanted into the injured area, and the expression and secretion of GDNFwere sustained, which solved the shortage of the short time of exogenous drug administration. Gene therapy should select suitable vector cells. Neural stem cells can proliferate in vitro and induce neural stem cells to differentiate into neuronal and glial cells. Therefore, NSCs have become the ideal vector cells for gene therapy. At the same time, chitosan has good compatibility with tissue, can be absorbed by tissue, can inhibit the growth of fibroblasts and prevent scar formation, so it is also an ideal material for the treatment of nerve injury. In this study, cell culture, gene transfection, flow cytometry, immunohistochemistry, HRP retrograde tracing, sciatic nerve function index, patch clamp electrophysiology, etc. The growth and differentiation of NSCs, chitosan and Schwann cells were observed. The GDNF gene was transfected into NSCs in vitro, screened, amplified and transplanted into the model of sciatic nerve transection by chitosan tube in order to satisfy the three factors of nerve regeneration and repair, and to observe and evaluate the therapeutic effect. To explore the new feasibility for the treatment of nerve injury. The main results are as follows: 1. In this experiment, NSCs were isolated and cultured in embryonic rat cerebral cortex. Cell suspension growth, cell cloning
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329;R318.08

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