CN106421912A - 一种基质化去细胞神经支架的制备和应用 - Google Patents
一种基质化去细胞神经支架的制备和应用 Download PDFInfo
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- CN106421912A CN106421912A CN201610905475.4A CN201610905475A CN106421912A CN 106421912 A CN106421912 A CN 106421912A CN 201610905475 A CN201610905475 A CN 201610905475A CN 106421912 A CN106421912 A CN 106421912A
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Abstract
一种基质化去细胞神经支架的制备和应用。利用天然猪视神经作为去细胞神经支架材料，制备一种具有天然多通道和孔道结构的支架。这种支架的纵行通道分布均匀、直径较均一，纵行通道之间含有横向孔道连接。在支架之中通过类细胞外基质改良，逆转这种天然生物支架材料细胞生长和营养因子贫瘠的微环境，为体外促进种植在其中的细胞存活、分化、迁移和形成神经网络提供支持，亦为其移植后促进受损伤脊髓上、下行神经纤维的重塑连接提供有利的微环境。
Description
所属技术领域
本发明专利涉及一种用于修复脊髓损伤的基质化去细胞视神经支架材料，尤其是一种能够通过类细胞外基质改良，逆转天然生物材料细胞生长和营养因子贫瘠的微环境，促进种子细胞在体外形成神经网络，移植后能够促进受损伤脊髓上、下行神经纤维重塑连接，通道和孔道分布及其直径较为均一的组织工程支架。
背景技术
在脊髓损伤的修复中，组织工程支架移植可以填充脊髓损伤处的空洞，为细胞和神经纤维的生长和攀爬提供支撑，有利于脊髓损伤结构和功能修复，因此，组织工程材料为脊髓损伤的研究提供了新的平台。就目前而言，脊髓损伤修复的理想解决方案是一种针对损伤机制的分子、细胞和组织水平的人工生物植入物，以生物可降解聚合物提供组织支架、细胞承载工具和缓释药物的存储等功能。生物材料和支架设计需要考虑的因素：1、生物相容性；2、适合组织定向整合和提供多孔性刺激的形态学上的引导索样结构；3、细胞相容性，促进最适宜的细胞黏附，迁移和轴突生长；4、物理特性(弹性力学、强度、韧性)与受体组织接近；5、生物降解后的产物无毒性。
到目前为止，针对脊髓损伤修复的支架大多只能提供神经纤维生长的环境支撑，神经纤维无法有序生长，因此无法高效准确的使上、下行神经纤维准确对接，治疗效果并不理想。在此基础上衍生出的高分子化合物支架，能够合成很好的纵行通道，促进神经纤维的对接。但是，聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid)，PLGA)等化合物的降解引起的酸堆积，都增进了损伤修复的负担。因此，寻找一种天然的无机体负担的原料，研发一种有明显纵行通道结构，并能介导神经纤维贴附其穿越生长的支架至关重要，支架内的纵行通道之间并不是封闭的，而是有横向孔道连通着，这有利于神经纤维之间的接触和信息传递。神经元轴突需要的通道和孔道直径约在几十微米到几百微米之间，目前工艺制备上难以制造出具有如此微小均一通道和孔道的组织工程神经支架材料。所以，我们尝试在天然生物组织中寻找合适的神经支架材料。
通常情况下，细胞之间填充着细胞外基质，它对细胞起着支持、保护和营养等作用，一般不具有明显的免疫原性。因此，目前在脊髓损伤以外的领域，利用细胞外基质来促进组织修复的方法在血管、皮肤、骨骼、软骨、肝脏、肺脏、心脏和外周神经等组织器官都展示出巨大潜力。然而，脊髓组织较松软，且细胞组分复杂，使得去细胞脊髓组织支架在脊髓损伤修复领域还未有成功案例。有研究表明，在体外，脱细胞中枢神经支架可以促进鸡胚绒毛膜的增殖迁移和神经细胞的分化，并且几乎不存在免疫原性。
有研究验证了几种中枢神经脱细胞的效果，这几种中枢神经分别是视神经，脊髓，脑组织，发现其细胞残留极少，保留了神经支持蛋白和生长因子，且可以与PC12细胞系相容。作为特化的脑神经，视神经有着特殊的结构，即有着明显的隔膜作为分区，并且横切面可见神经束分布均匀，直径多在100mm左右，较为均一。这与外周神经不同，坐骨神经的神经束直径较大，且直径相差很大。因此，去细胞视神经支架移植在脊髓损伤修复方面有着明显的优势，更接近于天然脊髓白质的组织结构，利于神经纤维成束形成和生长穿越。
神经干细胞(neural stem cells，NSCs)是指神经系统内未分化的、能自我增殖和自我更新，并具有向神经元和神经胶质细胞分化潜能的一群细胞。NSCs于1989年在小鼠的室管膜下层首次被证实，并于1992年首次从小鼠的纹状体组织和室管膜下层分离出来。NSCs的主要功能是作为机体神经组织一种后备细胞，替换正常死亡的细胞或参与神经组织损伤后的修复。现在可以利用NSCs具有自我更新能力和分化潜能的特性，将其移植治疗脊髓创伤性疾病。Park等人将PLGA与多聚赖氨酸制作的支架与NSCs移植到大脑损伤的小鼠后，发现其肢体运动功能恢复，并且支架本身也可以降低胶质疤痕的形成。之后，利用NSCs和施万细胞(Schwann cells，SCs)联合移植的方法也取得了良好的治疗效果，结果表明，这种方法促进了神经元轴突的髓鞘化，形成有利于轴突生长的微环境；NSCs移植入受损伤脊髓后能分化为神经元及神经胶质细胞，能减轻脊髓的继发性损伤，保护受损伤的神经元，促进肢体运动功能的恢复。
目前在国内外，一种天然多通道和孔道的基质化去细胞支架材料仍未见报道。为此，我们设想构建一种具有均一多通道和孔道的基质化去细胞猪视神经支架，同时利于这种支架构建神经网络样结构。拟将这种具有种植种子细胞的均一多通道和孔道生物活性支架移植入脊髓损伤处，连接脊髓损伤处再生的上、下行神经纤维，修复脊髓损伤。本发明的目的是想克服现有临床上治疗脊髓损伤的技术和方法上的不足，应用我们构建的具有均匀多通道和孔道的生物活性支架所构成的神经网络样结构来修复脊髓损伤。
发明内容
本发明专利提供一种基质化去细胞猪视神经支架材料，这种支架具有天然多通道和孔道结构，纵行通道之间含有横向孔道连接。该支架不仅能够促进种植的种子细胞形成具有信息传递功能的神经网络，且将其移植能够连接脊髓损伤处上、下行神经纤维发挥有效的神经元中继器作用。
本发明专利解决其技术问题所采用的技术方案是：
制备得到猪去细胞视神经支架后进行基质化改造，在支架中种植NSCs或背根节(DRG) 神经元，使NSCs分化为神经元，且其轴突沿通道和孔道生长，形成神经网络样结构，将这种神经网络样结构移植后可连接受损伤脊髓再生的上、下行神经纤维，修复脊髓损伤。
本发明专利的有益效果是：
这种多孔道基质化支架移植到损伤脊髓处后，其在体外已经形成的神经元中继器结构可介导上、下行神经纤维形成有序连接，即可连接损伤区域上、下行神经纤维一对一式的重新连接，在此基础上，去细胞视神经的孔道之间还存在一些连接通路，可以为新形成的神经纤维之间构成交叉对话。
附图说明
图1.示种植细胞后基质化的去细胞猪视神经支架的纵向示意图。(a示：猪去细胞视神经；1示：去细胞猪视神经形成的孔道；2示：神经元；2-1示：神经元胞体；2-2示：神经元轴突形成的神经纤维)
具体实施方式
下面通过具体实施例对本发明所用的主要仪器和试剂作详尽的描述：
1.主要仪器
超净工作台(苏州净化电子设备厂)；低速多管架自动平衡离心机(湖南湘仪实验室仪器开发有限公司)；5％CO2培养箱(Queue美国)；倒置相差显微镜(Olympus日本)；荧光显微镜(Leica德国)；扫描电镜(Philips荷兰)；低温烤箱(上海跃进医疗器械厂)；高温烤箱(上海跃进医疗器械厂)；高压消毒锅(江阴滨江医疗设备厂)；恒冷箱切片机(Shandon英国)；超纯水仪(Molsheim法国)；摇床(广州市正一科技有限公司)；冷冻真空抽干机(Labconco)
2.D-Hank’s平衡液(自配)，0.01mol/L PBS(中杉金桥)，Hoechst33342(Sigma)，山羊血清(中杉金桥)，脱氧胆酸钠(Sigma)，TritonX-100(上海生工)；β-NGF(Peprotech)；B27(GIBCO)；Matrigel(GIBCO)，DMEM/F12(Hyclone)，Neurobasal(GIBCO)。
本发明详细的具体操作技术说明如下：
1.基质化去细胞视神经支架的制备
从屠宰场取得新鲜的猪视神经，冰盒存放，立即将视神经周围多余的结缔组织及膜去除，置于-20℃冰箱中保存备用。
猪视神经脱细胞方法：剪去表面脂肪组织和部分神经鞘膜，将其截取为5mm的节段，置于蒸馏水中震荡，漂洗6h(60rpm)，将视神经放入30ml/L的Triton X-100(三硝基甲苯)水溶液中，并置于振荡器上震荡12h(60rpm)，在蒸馏水中漂洗3次，置于40g/L(4％)的脱氧胆酸钠水溶液中震荡24h(60rpm)，在蒸馏水中漂洗3次，重复3-6步2次，保存在PBS中，将去细胞视神经浸泡在4％多聚甲醛固定24小时，梯度蔗糖溶液脱水24小时。将固定好的去细胞视神经浸泡于75％酒精中15min，D-Hank’s溶液中漂洗3次，每次10min。在超净工作台上将消毒好的去细胞视神经材料放入已经高压灭菌过的冻干瓶中，在冻干机中冻干12h。将无菌的去细胞视神经放入基质混合物中，混合3h后交联。之后密闭保存在干燥皿中备用。
2.神经干细胞的种植
2.1神经干细胞(NSCs)的体外分离培养
选用出生1d的SD乳鼠2只，在无菌条件下取脑，将脑置于冷的D-Hank’s液中，解剖显微镜下用器械分离出海马。采用机械吹打法培养NSCs，先用眼科剪将海马组织剪碎，再连同D-Hank’s液移入离心管中用细头玻璃吸管轻轻吹打数次，直至肉眼见不到明显的组织块，吹打时应慢速并用力适度，避免产生气泡，以1000rpm离心5min，去上清，重复操作一次，用NSCs培养液重新吹打悬浮细胞沉淀，计数并调整细胞密度约为1×105个/ml，将此细胞悬液移入培养瓶，于37℃、5％CO2培养箱中进行悬浮培养。隔天进行半换液并轻轻吹打。
2.2神经干细胞的种植在超净工作台上将培养5d的神经干细胞吹入事先准备好的去细胞视神经中，加入Neurobasal培养液。培养7d，隔天换液。
3、背根节(DRG)的种植
3.1 DRG的体外分离
在解剖显微镜下将出生1d的带有绿色荧光蛋白基因(GFP)的新生大鼠的脊髓取出并置于一新的装有预冷DMEM培养基的培养皿中。(所有操作均在冰盒上进行)。用显微剪将DRG逐个剪下来放置于一个含有2ml预冷DMEM培养基的35mm培养皿中。尽量减少碎片的污染。
3.2 DRG的种植
在超净工作台上，于96孔板中放入直径3mm高3mm的PLGA膜做成的套管，并在板上铺Matrigel使之固定。将事先准备好的猪去细胞视神经放入套管中，切面向上。这时将取出的DRG放在去细胞视神经横切面上，小心加入Neurobasal培养液。于37℃、5％CO2培养箱中进行培养。培养7d，隔天进行换液。
4.去细胞视神经中细胞生长情况的检测
将培养7d的种植两种细胞的去细胞视神经分别使用冰冻切片机进行横切和纵切，厚度 为20μm，并在荧光显微镜下观察细胞生长状况。
实验结果显示：
1.应用细胞外基质和去细胞猪视神经构建成具有纵行多通道的天然支架
如图1显示所制备的基质化去细胞猪视神经支架的纵向形貌。去细胞猪视神经支架具有天然的纵行通道，纵行通道之间含有横向孔道连接。一方面，有利于种植于其中的神经元伸出长突起通过纵行通道与脊髓宿主的神经元接触；另一方面，有利于种植于其中的神经元之间通过横向的孔道彼此相连，交换上下纵行孔道间的信息。a示：去细胞猪视神经；1示：去细胞猪视神经形成的纵行通道；2示：神经元；2-1示：神经元胞体；2-2示：神经元轴突形成的神经纤维。
2.应用细胞外基质和去细胞猪视神经构建成具有天然分区的天然支架
基质化去细胞猪视神经支架的横向形貌显示，支架由圆形的纵行通道组成，纵行通道间可见横向的孔道连接，可用于细胞的分区种植。
3.去细胞猪视神经保留了视神经的三维圆柱形结构和天然的管道样结构
猪视神经和所制备的基质化去细胞猪视神经的HE染色显示，正常猪视神经横切面可见一个个圆形的管道，管道内充满神经纤维，神经纤维中可见大量苏木素着色的细胞核与伊红着色的细胞质和细胞外基质。纵切面则呈纵行管道样排列。去细胞猪视神经保留了神经鞘膜，有效的维持了去细胞支架的圆柱形三维空间结构，去除了通道中的神经纤维，横切面可见一圈圈由神经鞘膜伸入视神经内分隔形成的管道样结构，纵切可见一条条纵向的顺直通道组成，且通道间有横向孔道连接。
4.去细胞猪视神经DNA核酸残留检测
猪视神经和所制备的基质化去细胞猪视神经Hoechst33342染色显示，在荧光显微镜下猪视神经中含有大量蓝色的细胞核。而经过去细胞处理后，去细胞视神经支架中观察不到蓝色的细胞核，说明脱细胞的过程将细胞及胞内核成分均脱去，有效的避免了核酸残留对移植细胞造成的毒害和影响。
5.去细胞视神经的细胞相容性
带有GFP基因的DRG在去细胞猪视神经支架中种植7天显示，在荧光显微镜下绿色的DRG贴附在材料表面，材料中的绿色细胞和细胞突起沿着支架的纵行通道向内迁移生长，并且细胞间能通过横向孔道相互接触形成网络。这种天然的通道与孔道结构有利于细胞的长距离生长及信息交互。
带有GFP基因的NSCs在猪去细胞视神经支架中种植2天显示，在荧光显微镜下绿色的 NSCs种植在支架内，相对均匀的分布于由神经鞘膜伸入视神经内分隔形成的一个个圆柱形通道中，并通过通道间的横向孔道连接。这种由隔膜形成的分区有利于细胞的分区室种植，为支架材料中不同区域种植不同类型细胞奠定很好的结构基础。
Claims (4)
1.一种基质化去细胞神经支架的特征是：(1)取自于天然的猪视神经；(2)具有天然纵行通道，纵行通道之间含有横向孔道连接；(3)被负载不同的基质。
2.根据权利要求1所述的一种基质化去细胞神经支架的特征是：该支架是通过脱细胞方法制备获得，形成一种含有分布和直径较为均匀的通道和孔道的去细胞猪视神经支架。
3.根据权利要求1所述的一种基质化去细胞神经支架的特征是：被负载的基质应该是细胞生长和营养因子，如神经营养素-3(NT-3)、睫状神经营养因子(CNTF)、胶质细胞源性神经营养因子(GDNF)、脑源神经营养因子(BDNF)、成纤维细胞生长因子(FGF)、胰岛素样生长因子(IGF)或转化生长因子(TGF)中的一种或几种组合；应该是中枢神经或外周神经的细胞外基质中的一种或两者混合；亦应该含有具备细胞外基质分泌功能的细胞，如施万细胞或骨髓间充质干细胞。
4.根据权利要求1所述的一种基质化去细胞神经支架的特征是：能够将这种基质化去细胞神经支架制备成神经网络样结构，并将其移植到脊髓损伤处可修复受损伤脊髓的结构和功能。
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| CN110507859A (zh) * | 2018-05-22 | 2019-11-29 | 中山大学 | 含血管化区和轴突直行髓鞘化区的组织工程脊髓组织构建方法 |
| CN110584829A (zh) * | 2019-09-17 | 2019-12-20 | 深圳兰度生物材料有限公司 | 神经修复导管 |
| CN111093722A (zh) * | 2017-06-13 | 2020-05-01 | A·D·多斯塔 | 用于受损伤的神经组织修复的植入物、受损伤的神经组织的外科手术治疗方法和多孔聚四氟乙烯的应用 |
| CN115671028A (zh) * | 2022-11-11 | 2023-02-03 | 西北工业大学 | 一种基于plga的局部眼用缓释复合制剂及其制备方法和应用 |
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| CN109146075A (zh) * | 2018-05-02 | 2019-01-04 | 天津科技大学 | 一种用于提升骨髓干细胞活性的超声激励参数优化方法 |
| CN115518200B (zh) * | 2022-10-08 | 2024-01-26 | 中国医科大学附属口腔医院 | 一种脱细胞神经的制备方法及其脱细胞神经 |
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