CN110812532A - 一种靶向促进皮质脊髓束连接以修复脊髓损伤的组织工程支架的构建方法 - Google Patents
一种靶向促进皮质脊髓束连接以修复脊髓损伤的组织工程支架的构建方法 Download PDFInfo
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Abstract
一种靶向促进皮质脊髓束连接以修复脊髓损伤的组织工程支架的构建方法。运用PTPσ‑TrkC之间的配体和受体的相互结合的原理，将TrkC基因转染的神经干细胞种植到缓释NT‑3的多孔隙明胶海绵圆柱体支架，构建一个含过表达TrkC的干细胞源性神经元的三维生物支架，此支架移植到脊髓损伤区可以给表达PTPσ的再生的皮质脊髓束提供新的连接靶点，促进兴奋性突触的发育。这是一种可以靶向连接皮质脊髓束的组织工程支架的构建方法。
Description
所属技术领域
本发明专利涉及一种靶向促进皮质脊髓束连接以修复脊髓损伤的组织工程支架的构建方法。基于PTPσ-TrkC之间的配体和受体相互结合的原理，将TrkC基因转染的神经干细胞(neural stem cells，NSCs)种植到缓释NT-3的多孔隙明胶海绵圆柱体支架，构建一个含过表达TrkC的干细胞源性神经元三维生物支架。将此支架移植到脊髓损伤处，以此给再生的皮质脊髓束(corticospinal Tract，CST)提供新的连接靶点，是一种应用组织工程技术和转基因技术靶向连接CST从而治疗脊髓损伤的方法。
背景技术
脊髓损伤是一种致残率、死亡率都非常高的神经***疾病。据统计，世界范围内脊髓损伤每年发病率为23/百万，亚洲国家尤其中国和韩国发病率较高，为12.06-61.6/百万。目前我国约有200万脊髓损伤导致的截瘫患者，并且每年以约5万人的速度增加，而且呈现出高发病率、高致残率、低龄化的趋势，给家庭和社会带来了巨大的经济压力。仅在中国，因脊髓损伤导致的政府直接经济支出高达数十亿，因此脊髓损伤的研究与治疗成为世界各国研究的热点。
脊髓损伤后造成大脑和肢体之间的联系中断，因此大脑的信号无法到达损伤平面以下，导致病人损伤平面以下瘫痪，失去自主运动的能力。CST由大脑皮层的第五层大锥体神经元发出下行纤维组成，直接或间接地陆续终止于脊髓前角运动神经元，控制四肢和躯体的自主运动，CST被认为是恢复自主运动功能的关键要素。重新连接CST和脊髓尾端的信号通路，有望实现大脑信号对肢体的重新支配。关键科学问题是如何靶向连接CST，从而传递来自 CST的脑源性信号。移植NSCs可以实现部分连接功能，我们之前通过用三维明胶海绵支架和海马来源的NSCs在体外诱导培养，形成有突触连接的组织工程支架，将其移植到脊髓损伤处，可以实现与脊髓损伤区头端的固有神经元和尾端的固有神经元形成突触连接，并实现信号传导，促进动物行为功能的恢复[1-3]，在此基础上，如何使得移植干细胞靶向连接CST 的信号是尚未解决的关键问题。
有研究表明，与胚胎端脑或后脑来源的神经元相比，胚胎脊髓来源的NSCs分化的神经元，能更有效地促进脊髓损伤后CST再生并与其建立功能性突触，但是机制未明，推测可能与特异性的黏附分子的相互作用有关[4]。因此，CST轴突终末与新生的神经元之间的特异性黏附作用可能是提高二者之间的突触联系的关键因素。突触前膜上蛋白酪氨酸磷酸酶σ(protein Tyrosine Phosphataseσ，PTPσ)在调节CST轴突生长以及与靶位神经元建立突触过程中的作用倍受关注。有研究表明PTPσ可与突触后膜上神经营养素-3受体酪氨酸激酶C (TrkC)结合，从而提高突触前、后膜的黏着性能，促进兴奋性突触的发育[5]，而NT-3能进一步促进PTPσ-TrkC之间的粘合[6]。PTPσ是一种蛋白酪氨酸磷酸酶，在轴突上表达丰富，TrkC不仅仅是神经营养因子NT-3的受体，它还具有典型的细胞粘附结构域，胞外段是非催化亚单位，其可以通过绑定轴突前膜的PTPσ在兴奋性谷氨酸能突触中起重要作用[5]。因此，我们利用TrkC可与CST的轴突上的PTPσ结合的特性，将干细胞过表达TrkC后诱导分化为神经元，并移植到脊髓损伤区。从而达到提高突触前、后膜的黏着性能，促进兴奋性突触的发育，使得CST与外源性人工组织工程支架形成更多的突触连接的目的。
发明内容
为了克服现有脊髓损伤研究中CST的靶向连接不足的问题，实现传递来自CST的脑源性信号，本专利构建了一种含过表达TrkC的干细胞源性神经元的三维生物支架，其可以与再生的CST表达的PTPσ结合，通过特异性的黏附分子之间的相互作用促进更多的功能性突触的形成，从而促进脊髓损伤的修复。
本发明专利的基本方案包括：先构建过表达TrkC的慢病毒，转染海马来源的NSCs，再将其种植于缓释NT-3的多空隙明胶海绵圆柱体支架中，在体外构建一个外源性人工组织工程支架，再将这个人工组织工程支架移植到小鼠全横断脊髓损伤区缺失1mm的缺损区，利用 TrkC与PTPσ黏附分子之间的结合，促进再生的CST与移植的干细胞源性神经元形成突触，从而更好地连接CST。
本发明专利的有益效果：此方法通过特异性的黏附分子之间的相互作用，例如TrkC与 PTPσ的结合，靶向促进CST连接中继脑源性信号，从而促进脊髓损伤的修复，为临床上治疗严重的完全性脊髓损伤提供新的治疗策略。
附图说明
图1过表达TrkC的NSCs在三维支架薄片中与脑片共培养的连接情况。(图A示GFP脑片种植于过表达TrkC的NSCs中，bar＝40μm；图B示脑片来源的神经突起与过表达TrkC的细胞紧密接触，而这些突起表达PTPσ，bar＝10μm。)
图2体外培养的脑片来源的突起与过表达TrkC的NSCs源性神经元之间突触蛋白SYN 的表达情况。(图A示脑片与过表达TrkC的NSCs中SYN的表达情况，bar＝10μm；图B示脑片与转染空载病毒的NSCs中SYN的表达情况，bar＝10μm。图C为它们的统计图，P* p<0.05。)
图3总体思想模式图
图4载体图谱
具体实施方式
下面通过具体实施例对本发明所用动物信息、仪器、试剂和方法作详尽的描述：
1.动物信息
(2)小鼠：C57小鼠，由中山大学实验动物中心提供。
2.主要仪器和试剂
冰冻切片机(Thormo)，荧光显微镜(Leica)，0.01M PBS(中杉金桥)，Hoechst33342(Sigma)，山羊血清(GIBCO)，一抗(MAP2，SYN，Abcam)，二抗(Alex-555，Invitrogen)。
3.构建TrkC基因的慢病毒载体：
构建的此载体为带红色荧光蛋白报告基因并有嘌呤霉素抗性基因的过表达TrkC基因的慢病毒载体。
(1)设计引物序列和扩增各片段：
| Primer EF1A-TRKC-F | cagaacacaggaccggttctagagcgctgccaccATGGATGTCTCTCTTTGCCC |
| TRKC-MF | GCCCTCGTCACTGGATGCCGGGCCCGACACTGTGGTC |
| TRKC-MR | GACCACAGTGTCGGGCCCGGCATCCAGTGACGAGGGC |
| Primer TRKC CMV+ | CTACCTGGACATTCTTGGCTAGCGTTACATAACTTACGGTAAATG |
| Primer CMV TRKC- | CATTTACCGTAAGTTATGTAACGCTAGCCAAGAATGTCCAGGTAG |
| Primer RFP- | gagaagtttgttgcgccggatccCTTGTACAGCTCGTCCATGCC |
Lenti-puro-T2A-eRFP载体用XbaI和EcoRI双酶切，扩增四个片段，分别是TRKC、ⅠTRKC、ⅡCMV-GFP、CMV-RFP，采用重组克隆的方法，连接与转化，菌落PCR鉴定，测序，再进行质粒抽提。
片段扩增条件：
(2)质粒抽提步骤：
1).4000g离心收集菌液，弃上清，加10ml Solution1(加RNA酶),震荡混匀。
2).加10ml Solution2,轻轻颠倒混匀8-10次至澄清，室温静置2-3min，期间可颠倒混匀。 (注意这一步不能剧烈震荡，整个过程不要超过5min，Solution2用前溶解，用后拧紧盖，防止酸化)
3).加5ml冰浴N3，轻轻颠倒充分混匀10次至白色沉淀形成，静置2min，准备注射滤器，拔去活塞，置于50ml离心管上，将液体快速倒入注射滤器中。
4).轻轻塞进活塞将澄清裂解液过滤到离心管中，加入0.1倍体积的ETRSolution,颠倒混匀10次，冰浴10min，期间颠倒数次，再42℃孵育5min。
5).预处理HiBind DNAMaxi Column，用3mlGPS洗HiBind DNA Maxi Column,室温静置4min，25℃4000g离心5min，弃废液。
以下离心均为25℃
6).将第10步处理完的样品25℃4000g离心5min，将上清转移至新的50ml离心管，加 0.5倍体积的无水乙醇，轻轻颠倒混匀6-7次，室温孵育2min。
7).转移上一步得到的样10ml到HiBind DNAMaxi Column，4000g离心3min，弃废液，再加入上一步样，直至离完。
8).加10mlHBC buffer(加异丙醇)，4000g离心3min，弃废液。
(3)慢病毒包装、纯化浓缩及滴度测定
1)Day 0细胞接种：Low passage 293T接种于10cm/15 cm培养皿中(接种数量由预期的病毒量决定，1×10^8/15 cm培养皿)，控制接种密度，第二天生长至80％融合；
2)Day 1质粒转染：使用文库主质粒、psPAX2和pVSVG辅助质粒共同转染293T细胞；
3)Day 2换液：转染18小时后换液(转染体系有毒性),完全吸去培养基，小心加入20ml 新鲜完全培养基(15cm培养皿)；
4)病毒上清预处理：Day3，4，5收集病毒上清后，将培养上清转移至50ml离心管中，最高转速离心10min；，使用0.45um滤器过滤病毒上清，直接使用灭菌后的250ml离心瓶收集病毒上清；
5)高速离心纯化病毒：使用20ml注射器将10％蔗糖溶液，缓慢地注入离心瓶底部，体积比保持4:1(病毒上清4份，蔗糖溶液1份)，4度，14000rpm离心2小时；
6)病毒重悬：弃去上清，吸干净，加入100ul－1000ul PBS，吹吸混匀；
7)病毒上清分装，-80度冻存；分装时剩余30ul左右单独冻存，用于滴度测定。
8)使用化学发光法(CMIA)测定慢病毒滴度。
4.体外构建过表达TrkC的组织工程支架
(1)NSCs的培养选用出生1～3天的C57乳鼠4只，在无菌条件下断头取脑，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/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。当观察到大量的细胞克隆球开始形成时，隔天用细头玻璃吸管机械吹打分离NSCs克隆球进行传代。
(2)采用三维明胶海绵构建表达TrkC的组织工程支架选用P2代生长状态良好的NSCs，然后加入一定MOI值为5的带有嘌呤霉素抗性基因的RFP-TrkC慢病毒的新鲜培养液200μl，培养72h后，加嘌呤霉素筛选(终浓度为2μg/mL)，24h后离心弃去旧的上清液，补充新鲜培养液，再继续培养24h，将细胞种植于缓释NT-3的三维明胶海绵支架后，将支架放入含300μl 的10％FBS的DMEM/F12的24孔板中，每孔一个支架材料。于37℃、5％CO2培养箱培养14天，隔天更换培养液。
5.体外将脑片种植于过表达TrkC的NSCs上检测其与其中过表达TrkC的细胞形成连接的情况
选用出生1-3天的C57乳鼠1只，在无菌条件下断头取脑，将脑置于冷的D-Hank’s液中，解剖显微镜下用器械分离大脑皮层，去掉海马，将皮层切成200～500μm厚度的脑片，再分离成1mm左右的小块，轻轻置放在预先培养好的过表达TrkC的NSCs上，共培养7天，固定，染色，观察。
6.小鼠脊髓全横断模型的构建及体外构建的过表达TrkC的组织工程支架的移植
术前三天，小鼠皮下注射环孢素(0.3mg/10g)，术前腹腔内注射戊巴比妥钠(0.064mg/10 g)进行麻醉。经固定***、备皮消毒后，在无菌条件下切开皮肤、浅筋膜，用器械沿T8～T10 两侧棘突顺腰棘肌群走向钝性分离肌肉和韧带，用自制拉钩固定手术区域，清晰暴露T9棘突和椎弓，有齿镊轻提T9棘突，用眼科持针钳沿T9～T10椎弓间隙轻轻咬开椎弓根部，并逐渐咬下T9椎弓，暴露T10段脊髓。用直尖小梁剪剪开硬脊膜后，将一侧刀脚插到底部，快速全横断整个脊髓，在距头端横断处2mm再次横断脊髓，将中间的脊髓组织用显微镊小心取出，确保横断完全，充分止血后，再将体外构建的组织工程支架填塞入组织缺损区，依肌层、皮下组织、皮肤顺序逐层缝合。术后做好标记，每只动物肌肉注射青霉素16万单位1mL/d，连续3天，在膀胱区用手适度按压进行人工排尿，每日1～2次。为防止未长好的伤口被咬开，术后单笼喂养。此后，依据膀胱功能恢复情况，可逐渐减少排尿次数，动物饲养到时间点后取材检测，期间给以保温，自然光照时间，以及充分的饮食。
实验结果显示：
1.我们成功构建了TrkC的慢病毒载体，翻译后的氨基酸序列完全相同。测序结果如下：
5’-TCGGAGATGGATGTCTCTCTTTGCCCAGCCAAGTGTAGTTTCTGGCGGATTTTCTTGCTGGGAAGCGTCTGGCTGGACTATGTGGGCTCCGTGCTGGCTTGCCCTGCAAATTGTGTCTGCAGCAAGACTGAG ATCAATTGCCGGCGGCCGGACGATGGGAACCTCTTCCCCCTCCTGGAAGGGCAGGATTCAGGGAACA GCAATGGGAACGCCAGTATCAACATCACGGACATCTCAAGGAATATCACTTCCATACACATAGAGAAC TGGCGCAGTCTTCACACGCTCAACGCCGTGGACATGGAGCTCTACACCGGACTTCAAAAGCTGACCA TCAAGAACTCAGGACTTCGGAGCATTCAGCCCAGAGCCTTTGCCAAGAACCCCCATTTGCGTTATATA AACCTGTCAAGTAACCGGCTCACCACACTCTCGTGGCAGCTCTTCCAGACGCTGAGTCTTCGGGAAT TGCAGTTGGAGCAGAACTTTTTCAACTGCAGCTGTGACATCCGCTGGATGCAGCTCTGGCGGGAGCA GGGGGAGGCCAAGCTCAACAGCCAGAACCTCTACTGCATCAACGCTGATGGCTCCCAGCTTCCTCTC TTCCGCATGAACATCAGTCAGTGTGACCTTCCTGAGATCAGCGTGAGCCACGTCAACCTGACCGTACG AGAGGGTGACAATGCTGTTATCACTTGCAATGGCTCTGGATCACCCCTTCCTGATGTGGACTGGATAGTCACTGGGCTGCAGTCCATCAACACTCACCAGACCAATCTGAACTGGACCAATGTTCATGCCATCAAC TTGACGCTGGTGAATGTGACGAGTGAGGACAATGGCTTCACCCTGACGTGCATTGCAGAGAACGTGG TGGGCATGAGCAATGCCAGTGTTGCCCTCACTGTCTACTATCCCCCACGTGTGGTGAGCCTGGAGGAG CCTGAGCTGCGCCTGGAGCACTGCATCGAGTTTGTGGTGCGTGGCAACCCCCCACCAACGCTGCACT GGCTGCACAATGGGCAGCCTCTGCGGGAGTCCAAGATCATCCATGTGGAATACTACCAAGAGGGAGA GATTTCCGAGGGCTGCCTGCTCTTCAACAAGCCCACCCACTACAACAATGGCAACTATACCCTCATTG CCAAAAACCCACTGGGCACAGCCAACCAGACCATCAATGGCCACTTCCTCAAGGAGCCCTTTCCAGA GAGCACGGATAACTTTATCTTGTTTGACGAAGTGAGTCCCACACCTCCTATCACTGTGACCCACAAAC CAGAAGAAGACACTTTTGGGGTATCCATAGCAGTTGGACTTGCTGCTTTTGCCTGTGTCCTGTTGGTG GTTCTCTTCGTCATGATCAACAAATATGGTCGACGGTCCAAATTTGGAATGAAGGGTCCCGTGGCTGTCATCAGTGGTGAGGAGGACTCAGCCAGCCCACTGCACCACATCAACCACGGCATCACCACGCCCTCG TCACTGGATGCCGGGCCCGACACTGTGGTCATTGGCATGACTCGCATCCCTGTCATTGAGAACCCCCA GTACTTCCGTCAGGGACACAACTGCCACAAGCCGGACACGTATGTGCAGCACATTAAGAGGAGAGAC ATCGTGCTGAAGCGAGAACTGGGTGAGGGAGCCTTTGGAAAGGTCTTCCTGGCCGAGTGCTACAACC TCAGCCCGACCAAGGACAAGATGCTTGTGGCTGTGAAGGCCCTGAAGGATCACACCCTGGCTGCCCG GAAGGATTTCCAGAGGGAGGCCGAGCTGCTCACCAACCTGCAGCATGAGCACATTGTCAAGTTCTAT GGAGTGTGCGGCGATGGGGACCCCCTCATCATGGTCTTTGAATACATGAAGCATGGAGACCTGAATAA GTTCCTCAGGGCCCATGGGCCAGATGCAATGATCCTTGTGGATGGACAGCCACGCCAGGCCAAGGGT GAGCTGGGGCTCTCCCAAATGCTCCACATTGCCAGTCAGATCGCCTCGGGTATGGTGTACCTGGCCTC CCAGCACTTTGTGCACCGAGACCTGGCCACCAGGAACTGCCTGGTTGGAGCGAATCTGCTAGTGAAG ATTGGGGACTTCGGCATGTCCAGAGATGTCTACAGCACGGATTATTACAGGCTCTTTAATCCATCTGGA AATGATTTTTGTATATGGTGTGAGGTGGGAGGACACACCATGCTCCCCATTCGCTGGATGCCTCCTGAA AGCATCATGTACCGGAAGTTCACTACAGAGAGTGATGTATGGAGCTTCGGGGTGATCCTCTGGGAGAT CTTCACCTATGGAAAGCAGCCATGGTTCCAACTCTCAAACACGGAGGTCATTGAGTGCATTACCCAAG GTCGTGTTTTGGAGCGGCCCCGAGTCTGCCCCAAAGAGGTGTACGATGTCATGCTGGGGTGCTGGCA GAGGGAACCACAGCAGCGGTTGAACATCAAGGAGATCTACAAAATCCTCCATGCTTTGGGGAAGGCC ACCCCAATCTACCTGGACATTCTTGGCTAGTGGT-3’
2.在体外脑片与NSCs共培养体系中，观察到过表达TrkC能促进突触形成增加。
我们构建的TrkC基因的慢病毒载体可成功感染NSCs，并表达报告基因红色荧光蛋白，将转染红色荧光的TrkC的NSCs先种植在三维明胶海绵薄片中，培养7天后，再将绿色荧光鼠的脑皮质在显微镜下切成薄片，贴附在其上面，培养7天后固定染色，观察到脑片来源的表达PTPσ的GFP阳性神经突起与转染TrkC的NSCs紧密接触(图1)，并且在它们紧密接触的地方表达突触囊泡相关蛋白(synapsin1，SYN)，与空载病毒组相比，过表达TrkC组的 SYN的表达升高，统计结果显示两组有统计学差异，*p<0.05。(图2)。
参考文献：
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Claims (7)
1.一种靶向促进皮质脊髓束连接以修复脊髓损伤的组织工程支架的构建方法。其特征是：
(1)构建过表达TrkC的慢病毒载体；(2)用该病毒转染小鼠海马来源的神经干细胞(neural stem cells，NSCs)；(3)将过表达TrkC的NSCs种植于有自主知识产权的缓释NT-3的多孔隙明胶海绵圆柱体支架中，并在体外培养一段时间，构建一个人工组织工程支架；(4)将其移植到小鼠全横断脊髓损伤区；(5)该人工组织工程支架提供的TrkC可与皮质脊髓束(corticospinal Tract，CST)上的PTPσ结合，促进兴奋性突触的形成；
(6)通过黏附分子之间的相互作用促进CST与外源性人工组织工程支架更好的连接，传递脑源性信号。
2.根据权利要求1所述的构建的靶向促进皮质脊髓束连接以修复脊髓损伤的组织工程支架具有以下特征：(1)使用带TrkC的病毒载体转染细胞使细胞过表达TrkC；(2)运用组织工程的方法，种植该细胞到三维的生物可降解材料上，形成一个负载细胞的组织工程支架；(3)该支架体外培养诱导分化一定时间后，移植到脊髓损伤区可提供更多的TrkC靶点给CST上表达的PTPσ结合；(4)这种黏附分子间的特异性结合可促进兴奋性突触的发育，连接CST从而传递脑源性信号。
3.根据权利要求1所述的组织工程支架的特征，生物支架可为多孔隙明胶海绵圆柱体支架，或者其它天然、半合成或合成生物支架，例如胶原海绵，脱细胞的组织材料，壳聚糖，聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid，PLGA)，纳米微球等；提供NT-3的方式可以是通过负载NT-3在支架上做成缓释材料提供，也可以通过病毒转染细胞的方式提供，或者植入微泵持续释放外源性NT3的方式提供。
4.根据权利要求1所述的此种靶向促进CST连接的方法，其带TrkC的载体可以是慢病毒、腺病毒、腺相关病毒等各种病毒；或者是电转、脂质体转染等方法。
5.根据权利要求1所述的提供TrkC靶点的细胞可以是各种组织来源的成体干细胞(包括神经干细胞、间充质干细胞、脂肪干细胞、嗅鞘干细胞等等)、胚胎干细胞、以及诱导多能干细胞来源的各种干细胞或祖细胞，以及由它们分化来的细胞、或者幼稚或者成熟的神经元等。
6.根据权利要求1所述的此种靶向促进CST连接的方法，此方法可应用于促进包括人类，非人灵长类，犬类，猪类，羊类，兔类及鼠类等哺乳动物的脊髓损伤后皮质脊髓束的连接。
7.根据权利要求1所述的此种靶向促进CST连接的方法，除了TrkC和PTPσ这一对分子外，还可以是其他配对分子，例如蛋白激酶C(protein kinase Cγ，PKCγ)和N-甲基-D-天冬氨酸(N-methyl-D-aspartate receptor，NMDA)，整合素(integrin)和层黏连蛋白(laminin)，转录因子蛋白(T-box brain 1，Tbr1)和其受体Grin2b,信号素家族成员Sema3A和其受体neuropilin-1(NP-1)或受体复合物中的组分L1等，这些能给CST上表达的蛋白提供其配对的结合蛋白的方式。
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