促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后
Summary
含有生长因子的纤维蛋白基质被用来留住嫁接神经干细胞分化为完全脊髓横断网站。移植细胞完全充满病灶腔和分化为多种神经细胞类型，包括神经元轴突延伸到宿主脊髓长距离。
Abstract
神经干细胞（NSCs）能自我更新和分化成神经元和神经胶质细胞。移植的神经干细胞可以脊髓损伤（SCI）后替代丢失的神经元和神经胶质细胞，并能形成功能性继电器重新连接的上方和下方的病变脊髓节段。以前的研究接枝神经干细胞已经被不完全移植物存活的脊髓损伤腔内的限制。另外，接枝细胞存活，分化和扩展过程跟踪没有被优化。最后，在以往的研究，培养的大鼠神经干细胞通常报告分化成神经胶质细胞在移植到损伤脊髓，而不是神经元，除非命运被驱赶到一个特定的细胞类型。为了解决这些问题，我们开发了新的方法，以提高神经干细胞的存活，整合和分化，甚至严重的脊髓损伤部位。神经干细胞是新鲜从表达绿色佛罗里达州一个稳定的转基因菲舍尔344大鼠行胚胎第14天脊髓（E14）隔离uorescent蛋白（GFP），并嵌入到含有生长因子的纤维蛋白基质;该制剂的目的是保留移植细胞在病灶腔和支持细胞的生存。神经干细胞在纤维蛋白/生长因子鸡尾酒植入两周后胸3级（T3）完全性脊髓横断，从而避免炎症的高峰期。导致移植物完全充满病灶腔和分化为两个神经元，轴突延伸到宿主脊髓过相当长的距离，和神经胶质细胞。培养的人神经干细胞表达绿色荧光蛋白移植物导致类似的结果。因此，方法被定义为提高神经干细胞移植，存活和体内研究结果的分析。
Introduction
脊髓损伤（SCI）往往损害不仅白质束携带的信号和从脑，而且中央灰质，引起的interneurons和运动神经元的节段性损失。脊髓损伤的后果是两个马达和病变以下感觉功能的丧失。不幸的是，成人中枢神经系统（CNS）不自发地再生，从而产生永久功能缺损1。因此，重建受伤的成年脊髓和改进运动，感觉和植物神经功能是脊髓损伤研究的一个重要目标。神经干细胞（NSCs），无论是从胚胎或成年中枢神经系统中直接分离，是引人注目的候选细胞，以取代失去的神经元和神经胶质细胞。此外，这些细胞具有形成新的功能性继电器跨越病变部位2,3恢复轴突传导的潜力。
迄今为止，还没有解剖的详细阐释，electrophysiological和神经元中继形成了神经干细胞移植的重度脊髓损伤后行为的影响。有几个原因：第一，移植的神经干细胞或胎儿中枢神经系统组织中存活不佳时，嫁接到大病灶腔。以往的研究表明大量的早期细胞脱落，留下大空囊状病灶腔4,5。在一些研究中移植的细胞随后会分裂，并填写病灶腔4,5，但几天到几周的延迟之后这可能会发生，以及随后病灶部位填充可能不完整或一致的。其次，高效的跟踪系统，可提供对细胞存活，分化/成熟，并移植神经干细胞生长的声音数据缺乏。大多数早期的研究采用顺行和逆行标记来跟踪从移植2,3轴突的预测。然而，这些技术只是部分，往往从移植细胞所产生的标记不清轴突预测及示踪方法都是主观吨引起的染料泄漏以外植入的细胞的工件。其他组的人使用特定的神经元标记物来标记人类胎儿神经干细胞到受伤的啮齿类动物脊髓5,6移 植后轴突的预测。然而，在这些研究中， 移植并没有一贯良好的生存。最近，病毒递送的GFP报告基因的用于标记培养的神经干细胞7,8。但是，GFP的表达常常不一致，并且可以下调7。最近，利用转基因供体小鼠或大鼠稳定表达报告基因，绿色荧光蛋白，或人胎盘碱性磷酸酶，已显着提高体内 9,11移植的神经干细胞/祖细胞的跟踪。第三，一些研究表明，无论从胚胎或成年中枢神经系统导出体外培养的大鼠神经干细胞完全分化成神经胶质谱系当移植到完整的或受伤的成年脊髓心病的环境ð7,12,13，尽管事实是这些神经干细胞能够分化成神经元和神经胶质细胞在体外的，这表明局部环境可能要求干细胞的命运。可选地，培养的神经干细胞，尤其是那些从成年CNS衍生的，可具有固有defaulty属性分化为体内 13胶质谱系。
因为上面所讨论的限制，我们小组最近开发出一种新的协议，以提高胚胎NSC跟踪，生存，并在严重受伤的成年脊髓分化/成熟。简单地说，我们开始与表达绿色荧光蛋白报告基因在体内移植后14支撑GFP表达一个稳定的转基因菲舍尔344大鼠自交系。接下来，我们使用新鲜分离的神经干细胞从胚胎第14天菲舍尔344脊髓，发展阶段，其保存以产生神经元和神经胶质细胞的潜力。最后，我们的嵌入式新鲜分离的神经干细胞成含增长纤维蛋白基质因素15-17保留了细胞和均匀的大病灶腔内散发，旨在支持移植细胞的存活，分化和整合。移植物置入的T3完全横断，脊髓损伤后两星期网站。这些移植细胞始终充满完全横断网站和分化成神经元丰富的扩展大量的轴突到宿主脊髓长距离18。类似的结果用培养的人神经干细胞移植到免疫缺陷大鼠18获得的。
Protocol
Representative Results
Discussion
一个用于NSC移植在脊髓损伤的主要障碍是生存于病灶中心不佳。在病灶部位的任何间隙或腔体有可能减少或削弱棘上轴突和下方的伤害分离的脊髓节段之间的功能神经元中继的形成。另外，移植的神经干细胞存活不佳可能影响其与宿主组织整合，并因此减少移植神经元与神经元的主机的连通性。可能的机制来解释细胞的损失包括急性脊髓损伤产生的细胞毒性环境中，主机脊髓继发变性，受伤的区?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢鼠资源研究中心，美国密苏里大学，哥伦比亚大学，密苏里州，提供GFP鼠; Neuralstem公司提供人类神经干细胞。这项工作是由退伍军人管理局，美国国立卫生研究院（NS09881），加拿大脊髓研究组织，该克雷格H.尼尔森基金会，伯纳德和安妮·斯皮策慈善基金的支持。
Materials
| Reagents | Company | Catalogue | Comment |
| Fibrinogen(rat) | Sigma | F6755-25MG | 2 hr at 37 °C to dissovle Stock Concentration: 50 mg/ml Final Concentration: 25 mg/ml |
| Thrombin (rat) | Sigma | T5772-100UN | Dissovle in 10 mM CaCl2 Stock Concentration: 50 U/ml Final Concentration: 25 mg/ml |
| bFGF (human) | Sigma | F0291 (25 μg) | Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl |
| EGF (murine) | Sigma | E1257 (0.1 mg) | Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl |
| BDNF(human) | Peprotech | 452-02 (1 mg) | Stock Concentration: 1000 ng/μl Final Concentration: 50 ng/μl |
| NT-3 (human) | Peprotech | 452-03 (1 mg) | Stock Concentration: 1000 ng/μl Final Concentration: 50 ng/μl |
| GDNF (rat) | Sigma | G1401 (10 μg) | Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl |
| IGF-1(mouse) | Sigma | I8779 (50 μg) | Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl |
| aFGF (human) | Sigma | F5542 (25 μg) | Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl |
| PDGF-AA (human) | Sigma | P3076 (10 μg) | Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl |
| HGF (human) | Sigma | H9661 (5 μg) | Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl |
| MDL28170 | Sigma | M6690 (25 mg) | Stock in DMSO Stock Concentration: 1 mM Final Concentration: 50 μM |
| PBS | Millipore | BSS-1005-B | Stock Concentration: 1X Final Concentration: 1X |
| DMSO | Sigma | D2650 | |
| Ketamine | Putney | 26637-411-01 | 40-80 mg/kg Stock Concentration: 100 mg/ml Final Concentration: 25 mg/ml |
| Xylazine | Lloyd | 0410, | 2.5-8 mg/ml Stock Concentration: 100 mg/ml Final Concentration: 1.3 mg/ml |
| Acepromazine | Butler | 003845 | 0.5-4 mg/ml Stock Concentration: 10 mg/ml Final Concentration: 0.25 mg/ml |
| Betadine | Healthpets | BET16OZ | |
| Ringers | Abbott | 04860-04-10 | 2-3 ml/inj |
| Banamine | Schering-Plough | 0061-0851-03 | 2.5 -5 mg/kg Stock Concentration: 50 mg/ml Final Concentration: 0.5 mg/ml |
| Ampicillin | Sandoz | 0781-3404-85 | 80-100 mg/kg Final Concentration: 50 mg/ml |
| LH-RH | Sigma | L4513 | 200 μg/kg Final Concentration: 200 μg/ml |
| HBSS | Gibco | 14175-096 | |
| Trypsin | Gibco | 25200056 | Stock Concentration: 0.25 % Final Concentration: 0.125 % |
| DMEM | Gibco | 11995073 | |
| FBS | Gibco | 16000044 | Stock Concentration: 100 % Final Concentration: 10 % |
| Neurobasal Medium | Gibco | 21103049 | |
| B27 | Gibco | 17504044 | Stock Concentration: 100x Final Concentration: 1x |
Note, use human reagents for grafts of human NSCs |
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