Neurotrophic factors and spinal cord injury

doi:10.3969/j.issn.2095-4344.2013.02.033

Abstract

Abstract:

BACKGROUND: Neurotrophic factors can effectively suppress neuronal apoptosis and promote neural regeneration and differentiation after spinal cord injuries. But the short half-life, poor ability to pass through the blood-spinal cord-barrier and limitation for space reconstruction severely obstruct the applications of neurotrophic factors.
OBJECTIVE: To review the neurotrophic factor research progress and to find out more effective application methods providing assistance for both science and clinical research.
METHODS: Papers about neurotrophic factors for spinal cord injuries published from 2001-01 to 2011-12 in CNKI, WanFang, and PubMed databases were reviewed. “Neurotrophic factor; neurotrophin; spinal cord injuries; stem cell; bioengineer, tissue engineering” in Chinese and English served as the search term in the title and abstract retrieval. All the papers closely related to the neurotrophic factor applications were selected and those recently published or in the prestigious journals were selected in the same field. We got 379 initial papers and 41 were reviewed according to the inclusion criteria.
RESULTS AND CONCLUSION: A series of improvements and optimizations have been made for the deficient application of neurotrophic factors. Combined applications of neurotrophins to repair spinal cord injuries have a synergistic effect. Stem cells secrete neurotrophic factors continuously to help injured spinal cord reconstruction involved in stem cell differentiation by gene transfer technology. In addition, through bio-engineering techniques, hydrogel nanofiber scaffolds with neurotrophic factor are made to provide space and channels for neural pathway rebuild after spinal cord injuries. But the best combinations of neurotrophic factors, stem cells and biomaterials need further studies.

Key words: tissue construction, academic discussion in tissue construction, neurotrophic factor, spinal cord injuries, stem cells, carrier cells, bioengineering, tissue engineering, other grants-supported paper

CLC Number:

R318

Xu Wei, Cheng Li-ming. Neurotrophic factors and spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(2): 369-374.

Figures/Tables 1

2.1 纳入文献基本情况初检得到379篇文献，中文93篇，英文286篇。阅读标题和摘要进行初筛，排除因研究目的与此文无关及内容重复性的研究，共保存35篇中英文文献做进一步分析。文献[1-4]篇概括了神经营养因子家族的分类及受体类型，文献[7-16] 篇研究了神经营养因子的应用限制因素，文献[17-35]篇研究了神经营养因子与细胞生物学、生物工程、组织工程等联合应用修复脊髓损伤的相关研究进展。 2.2 结果描述 2.2.1 神经营养因子及其受体神经生长因子、脑源性神经营养因子、神经营养因子3、神经营养因子4/5组成人类4种主要神经营养因子[1]。近来研究还发现其他对神经有修复作用的小分子蛋白，如成纤维细胞生长因子家族，神经胶质细胞源性神经营养因子家族，睫状神经营养因子家族，胰岛素样生长因子I和II，变形生长因子β等。神经营养因子受体包括酪氨酸激酶受体家族(Trk受体家族)和P75神经营养因子受体。Trk受体3种亚型对神经营养因子具有一定的选择性，TrkA可以被神经生长因子与神经营养因子3特异性激活，TrkB对脑源性神经营养因子、神经营养因子4敏感，TrkC对神经营养因子3有较强的亲和力[2]。P75神经营养因子受体对细胞调控机制尚存在争议，早期认为其对神经营养因子前体的信号传导通路有介导作用，与Trk信号通路作用相反,可直接导致脊髓损伤后神经元细胞凋亡[3]。但也有证据表明P75对神经元凋亡与存活有双重调控作用[4]，有学者提出这可能与P75受体相结合的蛋白相关[5]，通过与不同的细胞因子组合诱导细胞的再生与凋亡，具体机制有待进一步深入研究，这些特点为脊髓损伤治疗提供了新的思路。 2.2.2 神经营养因子修复脊髓损伤应用障碍内源性神经营养因子：神经营养因子及其受体广泛存在于脊髓中，除神经营养因子3在脊髓发育早期含量较高外[6]，其他几类在脊髓中含量较低。脊髓损伤后神经生长因子在背根节中呈高表达状态[7]，Geng等[1]通过酶联免疫吸附法测得脑源性神经营养因子在脊髓损伤后24 h即出现增量表达，28 d后恢复至正常水平，具有时间相关性；TrkC蛋白在大鼠脊髓横断后7 d内呈下降趋势，7 d后开始上升，14 d后上升速度最为明显，与TrkC的mRNA表达曲线一致[8]。与Trk受体的上调方式不同，P75神经营养因子受体在脊髓损伤后的mRNA表达呈波动变化，并与神经细胞凋亡呈正相关，这种和神经元凋亡相关的波动变化与脊髓损伤后的调节与修复有重大关系[9]。脊髓损伤后神经营养因子及其受体的上调具有时间相关性，并具有不同程度的促进脊髓修复作用，但由于其上调程度有限，导致其促进修复和神经功能恢复作用有限并无法长期维持有效的治疗浓度，因此广大学者尝试利用外源性神经营养因子进一步增强脊髓损伤修复。外源性神经营养因子：外源性神经营养因子可通过静脉、腹腔、肌肉及皮下等到达脊髓，但由于此途径给药血浆中神经营养因子浓度下降快，另外神经营养因子的分子特性决定了其难以通过血脑屏障和/或血脊髓屏障[10]，所以经过体液稀释及血脊髓屏障的屏障作用后到达受损部位极其微量，难以发挥神经保护作用；神经营养因子也可以直接注射于伤区局部、蛛网膜下腔等发挥作用。损伤区局部给药有利于局部形成高浓度，有利于神经元的存活，由于神经营养因子的半衰期较短[11]，无法持续作用于损伤脊髓，针对这一情况，有部分学者采用微泵法给药[12]，但微泵寿命短，价格贵，植入及取出有创，存在感染等风险较大，一般只用于短期动物实验。在脊髓损伤后对神经营养因子的需求具有时间相关性，神经营养因子衰期较短的生物学特性导致单纯一种神经营养因子已经无法满足脊髓损伤修复需要。虽然外源性神经营养因子能够有效抑制神经元细胞凋亡，但损伤局部中心区域由于细胞坏死、凋亡，单纯营养残存的神经元无法满足神经通路重建需要，此外，大量的脊髓空洞形成，神经轴突无法通过缺损空间完成重建[13]。基于神经营养因子以上的应用障碍衍生不同应用策略。 2.2.3 神经营养因子修复脊髓损应用策略多因子联合：为满足脊髓损伤修复需要，不同神经营养因子间的联合或与其他小分子的联合修复脊髓损伤是近年来的研究热点。早期研究认为甲强龙具有神经保护作用，但近期Aomar等[14]的回顾性研究认为其并无神经症状的改善作用,但Kim等[15]将脑源性神经营养因子与甲强龙联合治疗脊髓损伤，并检测到髓鞘再生，结合免疫组化证实联合治疗的可行与有效性。Arvanian等[16]利用出生后第2天大鼠制作半切脊髓损伤模型，予以神经营养因子3与麦角酸二乙基酰胺联合治疗，发现与单纯利用神经营养因子3与麦角酸二乙基酰胺对照组相比，联合组有更早的行为学恢复时间与更好的恢复程度。随着对神经营养因子认识的增加，有学者尝试将多种因子联合，发现其治疗脊髓损伤有更好的疗效。Sharma等[17]研究中心在研究脊髓损伤后应用高浓度(0.5 mg)脑源性神经营养因子和神经胶质细胞源性神经营养因子，发现运动功能恢复及抑制血脊髓屏障破坏有明显改善，并减轻水肿，从而达到抑制神经元凋亡与促进再生作用。但有些组合无法发挥相应功能甚至出现拮抗作用，Lang等[18]发现脑源性神经营养因子和睫状神经营养因子的组合在神经根撕脱模型中未表现出协同作用，Donnelly等[19]也发现小干扰RNA与神经营养因子3联合治疗脊髓损伤未表现出更好的治疗效果。神经营养因子的协同作用增强了脊髓损伤修复，但如何完善神经营养因子及其他小分子组合方案有待进一步研究。干细胞辅助：神经营养因子能够促进干细胞再生分化，所以Cao等[20]将睫状神经营养因子与少突胶质细胞前体细胞联合治疗大鼠胸髓损伤，损伤区的少突胶质细胞前体细胞是对照组的4倍，这为少突胶质细胞前体细胞的进一步分化诱导奠定了基础。Deng等[21]将神经胶质细胞源性神经营养因子与Schwann细胞植入横断大刀鼠脊髓后发现胶质细胞迁移增强，胶质纤维酸性蛋白和硫酸软骨素蛋白多糖降低，为再生神经元创造再生空间方向。Kusano等[22]用神经营养因子3还可以和神经祖细胞构建分泌型神经营养因子3/D15A，不仅对脊髓损伤急性期有治疗作用，对慢性期也能够增加髓磷酯形成，改善大鼠后肢功能。由于神经营养因子的半衰期较短，与干细胞联合时代谢较快，无法持续作用于损伤脊髓，针对这一情况，有学者用神经营养因子基因转染干细胞，使其持续分泌，提高干细胞存活并参与诱导分化。Zhang 等[23]利用神经营养因子3基因转染神经干细胞移植治疗急性脊髓损伤，1月后在损伤区检测到大量的神经营养因子3表达载体，并通过血脑屏障评分证明大鼠运动功能恢复。Brock等[24]在成年恒河猴脊髓半切损伤后，将脑源性神经营养因子与神经营养因子3基因转染的成纤维细胞植入受损部位，移植8个月后发现神经元凋亡数目为对照组的1/4，并发现脑源性神经营养因子不仅对损伤区局部神经元凋亡有抑制作用，对远端神经元也有促进分化与轴突再生功能。近年来发现骨髓间充质干细胞因其良好的生物相容性，出色的免疫调节及免疫规避能力等成为承载神经营养因子基因的良好载体[25]。Koda等[26]用脑源性神经营养因子基因转染骨髓间充质干细胞后移植治疗大鼠完全横断脊髓损伤，6周后发现脑源性神经营养因子基因转染移植组大鼠后肢功能明显恢复，而单纯利用脑源性神经营养因子却未发现大鼠后肢运动功能的改善。Zhang等[27]用腺病毒将神经营养因子3基因转染骨髓间充质干细胞，并在维甲酸的诱导下分化成为神经元，并作为治疗脊髓损伤潜在细胞移植治疗脊髓横断大鼠，相较单独注射骨髓间充质干细胞组，骨髓间充质干细胞-神经营养因子3组脊髓空洞减少，损伤区神经元存活率升高，大鼠行为学评价改善，人微管相关蛋白2阳性的神经元向损伤区渗入，但在大鼠体内骨髓间充质干细胞向神经元的后期分化很轻微。神经营养因子的基因转染干细胞，使其在干细胞的分化过程中持续分泌，不仅保护受损部位残存的神经元，同时促进干细胞分化，但基因转染细胞治疗的稳定性、安全性有待进一步改进。生物工程改造：脊髓损伤后胶质瘢痕与脊髓空洞的形成严重阻碍脊髓神经元细胞的再生。脊髓损伤后为了规范神经轴突的生长方向并提供生长空间通道，Gros等[28]用琼脂糖结合神经营养因子3制作了模板化支架，但宿主-支架交汇处限制了再生轴突宿主脊髓的联系，使再生神经元轴突很难突破交汇区，建立有效的神经再生通路。为了局部形成有效的治疗浓度，延长神经营养因子半衰期，Burdick等[29]用聚乙烯制作了可降解水凝胶，通过控制交联密度来控制睫状神经营养因子的缓释速率，调控神经营养因子持续作用时间，持续至损伤后数周到数月。为改善水凝胶造成损伤区占位，Piantino等[30]利用高分子聚合物发明了可用于注射的液体水凝胶，不仅自身能够促进损伤区轴突再生，用其联合神经营养因子3缓释治疗脊髓损伤，较单用水凝胶组取得行为学评价方面进步。Tsai等[31]将这一可降解水凝胶合成弹性模量为(263±13) kPa的水凝胶通道(pHEMA-MMA)，将水凝胶通道植入并将通道内部填充神经营养因子3，辅以胶原、纤维蛋白、人工基底膜、甲基纤维素等，植入胸髓完全横断SD大鼠模型中，8周后观察到神经营养因子3明显降低了神经元凋亡数量，并促进神经元的再生，引起运动功能改善，这种复合的水凝胶通道对于完全横断脊髓有较好的修复作用。近年来还有学者用纤维蛋白原支架将神经营养因子3，血小板源性生长因子和/或肝素相关释放系统封装，促进脊髓损伤亚急性损伤区域神经元存活与再生[32]。生物工程具有结构和化学多向性，神经营养因子联合生物工程可以填充受损部位，为脊髓损伤修复提供空间通道，并将神经营养因子局部富集，利用交联密度缓释，减少胶质瘢痕，达到更好脊髓修复作用，但如何增强轴突穿透力与交汇区通透性，如何在众多材料与神经营养因子中找到最佳的组合仍需进一步探索。其他：针对神经营养因子难以穿透血脊髓屏障，因此金大地等[33]用分子克隆方法构建表达载体pTAT-HA-脑源性神经营养因子，研究合成的新型神经生长因子TAT-脑源性神经营养因子融合蛋白，通过尾静脉注射证实其兼具穿越血脑屏障及神经保护双重活性，为全身给药提供了新的思路。此外有研究证实电针刺激法可以激活脊髓神经元细胞Ca2+-Erk介导信号传导通路从而提高脑源性神经营养因子的表达，为增加内源性神经营养因子提供参考[34]。近来有学者报道一些具有神经营养因子活性的小分子，如7,8-二羟黄酮等对TrkB受体有靶向激活作用[35]，但仍处于研究阶段，有待进一步探索。"

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