【作者】 曹莉；

【导师】 路长林；

【作者基本信息】 第二军医大学 ， 神经生物学， 2002， 博士

【摘要】 脊髓损伤导致损伤平面以下截瘫是世界医学难题，目前仍无有效的治疗方法。遗憾的是哺乳动物中枢神经系统（CNS）轴突损伤后不能正常再生。目前认为，应用细胞移植、外源行给予神经营养因子等多种方法改善损伤局部的微环境，可以促进损伤的脊髓神经纤维再生。由于各治疗方法对神经组织的作用方式不同，其中两者联用可能产生超过单一因素的作用。因此，多种治疗方法的联合使用是今后治疗脊髓损伤的重要方向。 近年来，由于嗅鞘细胞（Olfactory ensheathing cells，OECs）对中枢神经再生有明显的促进作用，受到研究人员的高度重视，被越来越广泛地用作细胞移植治疗中枢神经系统损伤。嗅鞘细胞与雪旺氏细胞具有相似的特点，而其优点在于它可以通过周围-中枢神经移行区，并可以存在于中枢环境。这一独特的细胞学特性可以使CNS再生潜力得以发挥，己发现移植在中枢神经损伤处的OECs能形成细胞桥引导神经突起生长，并向很远距离延伸，使中枢神经损伤得以修复。 胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor，GDNF）是目前发现的在体外支持运动神经元生长最有效的神经营养因子，它不仅能挽救发育过程中运动神经元的程序性死亡，还可以促进脊髓损伤后皮质神经元的存活。在离体实验和在体坐骨神经切断后，均观察到GDNF对脊髓运动神经元具有明显的保护作用。 本研究首先应用原代培养技术，体外培养纯化OECs。根据p75NGFR免疫细胞荧光染色及Hoechst细胞核标记染色分析了所得细胞的纯度。并对不同培养时期的细胞活性进行比较，优选出一种较好的纯化方案。然后建立成年SD大鼠脊髓T₈半横断损伤模型，将体外培养纯化的OECs植入脊髓损伤腔，同时局部应用GDNF。采用斜板试验和 11 中英文摘要 BBB联合功能评分观察大鼠运动功能恢复情况，并通过辣根过氧化物 酶逆行示踪技术评价OECS和GDNF对神经元存活和纤维再生的影响。＿ 研究进一步通过研究GDNF对体外纯化培养OECs存活和增殖的影响－ 及其作用的信号传导通路，分析OECs与GDNF对脊髓损伤协同作用 的机理。最后利用重组逆转录病毒构建了高度表达GDNF 的OECS一＿ GDNF细胞株。从轴突再生、神经元保护作用和行为学实验三个角度研， 究，发现OECS—GDNF 细胞对脊髓损伤后的修复作用明显强于天然－ OEC‘移植。＿ 本研究主要结果如下＊ 一、成功建立OECS体外原代培养和纯化方案， 应用原代培养技术，并用差速贴壁、阿糖胞昔抑制和 Forskolin、＿ BPE营养物处理，分别从新生13d及2．5月成年大鼠的嗅球分离、培。 养和纯化OECs。根据p75 NGFR免疫细胞荧光染色及Hoechst细胞核－ 标记染色分析了所得细胞的纯度。并对不同培养时期的细胞活性进行。 ．呸瞩 比较。结果发现新生大鼠来源的OECs 纯化周期为sd，所得纯度为 80－85％，纯化后 18－20d左右细胞活力开始下降；成年大鼠来源的 OECS， 纯化周期为 10d，所得纯度可达 95％以上，纯化后 12－14d左右细胞活 力开始下降。研究表明新生大鼠来源的OECS优点为活力较高，取材方。 便，但成年大鼠来源的OECS所得纯度较高，更利于进一步研究。 二、0丑CS和＊＊＊F对大鼠脊髓损伤后神经元的保护作用 建立成年SD大鼠脊髓T。半横断损伤模型，将体外培养纯化的OECS 植入脊髓损伤腔，同时局部应用GDNF。按治疗手段不同，将实验大鼠 分为对照组、GDNF组、OECs组和 OECs＋GDNF联合应用组。8周后， 通过计算皮质和红核损伤侧神经元平均面积评价OECS和 GDNF对大 脑皮层神经元和中脑红核的逆行性保护作用。结果表明，与正常侧大 鼠皮质、红核神经元大小相比，对照组损伤侧神经元明显萎缩；GDNF 组皮质神经元比对照组明显增大，红核中两者虽无统计学差异，但 GDNF组大神经元数目（600pm‘以上）多于对照组；OECs组的皮质和 3 11 中英文摘要 红核神经元均比GDNF组明显增大。本实验表明局部应用外源性OECS 和GDNF均能减轻脊髓半横断损伤后引起的逆行性神经元损害。 三、OECS和GDNF大鼠脊髓损伤后轴突再生和后肢运动功能的影响 在上述损伤模型及实验分组的基础上，采用斜板试验和 BBB联合 功能评分观察大鼠运动功能恢复情况，并通过辣根过氧?更多还原

【Abstract】 Paraplegia below the level of injury resulted from traumatic spinal cord injury (SCI) is one of the tough problems in the world, which is still lack of effective method in clinical treatment. Unfortunately, injured axons in the mammalian central nervous system (CNS) do not regenerate normally. In recent years, substantial evidence has suggested that the failure of injured axons to regenerate within the mature central nervous system (CNS) can be overcome by providing growth-permissive environment at the injury site. Several strategies have been developed to achieve this goal including adding growth factors and transplanting functional cells. Since little of these strategies alone have been sufficient in repair of adult CNS, two or more strategies might be used in combination to obtain adequate structural and functional rebuilt.Potential therapeutic role of olfactory ensheathing cells (OECs) for repair of spinal cord injuries has been highlighted by recent reports. OECs are thought to play a key role in promoting and guiding the olfactory axons regenerate into CNS. In recent years, many studies have shown that transplants of OECs into lesions in spinal cord are also able to stimulate the growth of axons and in some cases restore functional connections.Glial cell line-derived neurotrophic factor (GDNF), a transforming growth factor (TGF)-P super-family trophic factor for midbrain dopaminergic neurons, has been found to support the survival of several neuronal cells including cultured spinal motoneurons and sensory neurons. GDNF is the most potent motor neuron trophic factor among NTFs found so far. GDNF might be useful in the treatment of neurodegenerative diseases and nerve injuries.In present paper, primary cultures of olfactory ensheathing cells (OECs) were dissociated from new born and 2.5 month old rat firstly. Purification of OECs was developed with cytosine arabinoside (Ara-C) treatment and differential adhesion method. OECs were stimulated to propagate by BPE or forskolin. The purity of OECswas determined according to immunostaining positively for p75 at different time. Viability and purity of OECs at distinct time was observed and compared. Adult OECs culture was selected for further experiment. Secondly, adult SD rats receiving Tg spinal cord hemisection were treated with GDNF and/or OECs, and the ability to promote axon regeneration and prevent neuron atrophy after spinal cord injury (SCI) was investigated. Animals were behaviorally tested for 8 weeks using the Basso, Beattie, Bresnahan locomotor rating scale and inclined plane test. At the end of 8 weeks, horseradish (HRP) labeled rubrospinal neurons (RSN) and corticospinal neurons (CSN) were counted and the mean soma size of RSN and CSN were quantified. Finally, the survival promoting effect and the possible intracellular signal transduction way of GDNF on OECs culture were examed to further investigate the mechanism of synergistic effect of GDNF and OECs.The main results of our research are as follows:1. Primary cultured OECs with high purityPrimary cultures of OECs were dissociated from new born and 2.5 month old rat. Purification of OECs was developed with cytosine arabinoside (Ara-C) treatment and differential adhesion method. OECs were stimulated to proliferate by BPE or forskolin. The purity of OECs was determined according to immunostaining positively for p75 at different time. Furthermore, viability of OECs with distinct derivation was observed and compared. The result demonstrated that the time for purification of OECs derived from new born rats was 5d with 80-85% purity, and the cell viability decreased about 18-20 days after purification. Meanwhile, total purification time of OECs derived from adult rats was 10d with more than 95% purity, and cell viability decreased about 12-14 days after purification. Adult OECs culture was selected for our experiment because of the high purity.2. Neuronal protection effects of GNDF and OECs after SCISD rats receiving T8 spinal cord hemisection were divided into 4 groups: cont更多还原