The application of beta-sheet self-assembling peptide hydrogels in neural tissue engineering

doi:10.3969/j.issn.2095-4344.0720

Abstract

Abstract:

BACKGROUND: Self-assembling peptides, which allow for an optimal control over the scaffold structure and composition, are described as the innovative tissue-engineered scaffolds in the field of biological medicine.

OBJECTIVE: To summarize the biological features of β-sheet self-assembling peptides and current research progress in the neural tissue engineering.

METHODS: We took “β sheet, self-assembling peptide, hydrogel, neural stem cell, neural tissue engineering” as the key words in Chinese and English, respectively, to retrieve the related literatures (2000-2017) from PubMed, CNKI, WanFang databases based on internet search.

RESULTS AND CONCLUSION: β-sheet self-assembling peptide, designed to function as an engineered mimic of the extra-cellular matrix (ECM), may be used with as a medium to direct growth and axonal connection, promoting neural stem cells adhesion, migration, division, outgrowth, neurogenesis and synaptogenesis. The self-assembling peptides RADA16, LDLK12, QL6 and their functionalized sequences have been proven to be of good utility. As the excellent performance of inducing peripheral nerve regeneration, mediating spinal cord injury and attenuating neuroinflammation, β-sheet self-assembling peptides have promising applications in the neural tissue engineering.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Protein Folding, Hydrogel, Neural Stem Cells, Tissue Engineering

CLC Number:

R318

Wei Wei, Liu Yan-fei, He Yang, Zhang Ling. The application of beta-sheet self-assembling peptide hydrogels in neural tissue engineering[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(10): 1586-1592.

Figures/Tables 2

短肽分子还可通过亲水和疏水区域的相互作用形成不同的聚合体结构。调整短肽中疏水或亲水性氨基酸的种类、数量和位置，就可得到具有不同形貌和理化特性的组装体。 2.1.2 β折叠二级结构自组装短肽有多种二级机构，包括α螺旋、β折叠、β发夹和卷曲螺旋等，其中β折叠结构是最常见且应用最广泛的二级结构。一般来讲，疏水性氨基酸与亲水性氨基酸周期性排列决定了β折叠的结构特性，随后通过分子内疏水性界面间的疏水作用力和分子内亲水性界面间的静电作用力共同堆积形成纳米纤维。由于驱动β折叠自组装的分子内作用力十分强劲，因此水凝胶可在体外培养环境和体内移植物中稳定存在。这些水凝胶具有自我修复能力，通过使用超声波处理破坏分子间作用力，促使水凝胶瓦解为液体状态，随着时间推移，短肽分子会逐渐重组装为与原先结构相同的纳米纤维。重组装的水凝胶机械强度等力学特性未发生太大改变。通过计算分析表明，由于氢键较好的定向连接，反平行β折叠比平行结构键能更高[10]。肽间及链间的非共价键作用增加了自组装短肽水凝胶支架的硬度。那些疏水与亲水氨基酸相互交替较多的短肽通常更容易形成β折叠，并且自组装成为超分子结构。 2.2 β折叠型自组装短肽在神经组织工程中的应用见表1。 2.2.1 对干细胞的促分化作用神经干细胞的生存、分化和增殖受细胞外基质成分、纳米拓扑结构和机械应力传导特性等的调控，而这些条件可在自组装短肽水凝胶支架中被部分重现[11]。自组装短肽水凝胶支架包含长度为5-200 nm的微孔，这种微孔尺寸与已发现的天然神经细胞外基质结构类似，细胞可迁移和黏附其中，为神经干细胞的迁移、分化和增殖提供一个极佳的基质环境[12]。体外二维环境中，神经干细胞的分化研究已取得了显著进步，但目前在三维环境中对神经干细胞的定向分化研究较少。研究发现，在三维环境中至少有2个关键变量——机械应力和生物活性，会影响神经干细胞的分化命运。神经干细胞对周围生长环境十分敏感，在刚性较强的三维基质中倾向于分化为星形胶质细胞，而在较软的基质中则倾向于分化为神经元细胞[13]。因为RADA16与促进细胞黏附的纤连蛋白、层粘连蛋白和胶原蛋白的序列RGD相似，在RADA16组成三维水凝胶支架微环境中的β-Tubulin、GFAP和Nestin阳性细胞数量与人工基质胶中的相似，结果显示它们对神经干细的分化有促进作用[14]。经典的RADA16是最具有代表性的β折叠型自组装短肽材料，拥有良好的成胶性，但溶于水后显示出其较低的pH值(3.0-4.0)，易引起组织损伤，且生物活性并不理想。因此，研究人员从一系列自然活性肽中挑选出不同的功能序列与β折叠型短肽连接，设计出功能丰富的水凝胶短肽。这些短肽既保持了良好的成胶特性，又显示出一定的生物学活性。如Cheng等[15]将层粘连蛋白衍生肽IKVAV序列连接于RADA16末端，得到功能化的β折叠型自组装短肽，形成的RADA16-IKVAV水凝胶支架与大脑组织的机械强度相似，可增强神经干细胞的黏附及向神经元细胞分化的能力。IKVAV序列模体并没有诱导分化，而是通过影响神经生长因子来增强分化，随着自组装短肽水凝胶支架硬度的增加，神经元细胞的密度下降而星形胶质细胞数量增加。Cunha等[12]采用RGD序列和BMHP(来源于骨髓归巢肽)序列分别修饰RADA16，研究了小鼠神经干细胞的三维分化，结果发现加入功能化序列的水凝胶支架增强了神经干细胞的生存、增殖和分化能力。空间中高密度的功能化序列可能是提高神经干细胞分化效果的关键。Luo等[16]通过腺病毒载体将人脑源性神经营养因子基因导入大鼠骨髓间充质干细胞中，再将其种入不同的水凝胶支架中，结果显示RADA16-PRG(PRG中包含RGD序列)水凝胶组的细胞生长、分化、神经营养因子蛋白和神经特异性烯醇酶表达均明显高于RADA16水凝胶组，证明在自组装短肽水凝胶中，骨髓间充质干细胞可通过人脑源性神经营养因子基因修饰分化为神经细胞。Sahab Negah等[17]设计的RADA4-GGS-IKVAV短肽，可促进神经干细胞的生存、黏附、分化和迁移，并且可减少神经干细胞向神经胶质细胞分化。Wang等[18]设计了RADA16-FGL水凝胶支架，可形成较弱的β折叠结构，与RADA16混合后自组装为纳米纤维体结构，不仅对大鼠脊髓神经干细胞无细胞毒性，还可促进干细胞的增殖及其向三维支架中迁移。 Caprini等[19]从调节干细胞增殖和分化的蛋白中选出短肽序列KLPGWSG，与 LDLK12自组装短肽结合，设计出新型功能化短肽支架，可明显增强神经干细胞的神经元分化。Gelain等[20]将FAQRVPP序列连接于LDLK12得到功能化的Ac-FAQ-LDLK12水凝胶支架，可促进人源和鼠源神经干细胞向神经细胞和少突胶质细胞分化。Koutsopoulos等[9]通过对比实验证明了相较于人工基底膜和Ⅰ型胶原蛋白培养系统，β折叠自组装短肽水凝胶支架明显促进了神经干细胞的神经元向分化，提高了细胞长期生存率。Shi等[21]将人脐带间充质干细胞和活化的星形胶质细胞共同种入RADA16-脑源性神经营养因子短肽水凝胶支架，该三维支架可促进具有神经轴突的典型神经元样细胞分化。此外，结果显示共培养体系会促进更多脑源性神经营养因子的分泌，有助于外源性人脐带间充质干细胞的神经元分化和内源性神经再生。 2.2.2 治疗神经退行性疾病阿尔茨海默病的神经病理学特征，包括细胞外β淀粉样蛋白斑块形成及神经元的减少。Cui等[22]将神经干细胞植入RADA16-YIGSR支架，发现该自组装短肽可自发性地组装为纳米纤维，它不仅可在普通培养条件下促进细胞生存率，还可减少由β淀粉样蛋白诱导的凋亡细胞数量。与单独使用神经干细胞相比，种植于自组装短肽水凝胶中的神经干细胞能表现出更多的神经元向分化。在阿尔茨海默病模型中，神经干细胞联合自组装短肽治疗对阿尔茨海默病模型大鼠学习、认知和记忆功能方面都有显著改善。因此，β折叠自组装短肽生物材料可优化神经干细胞移植治疗阿尔茨海默病的疗效，通过改善移植干细胞的生存和分化能力，促进神经保护效应，发挥抗神经炎症作用和旁分泌作用。帕金森病是一种以黑质多巴胺能神经元选择性变性为主要病理改变的神经系统退行性疾病。Ni等[23]发现，自组装短肽水凝胶可为多巴胺能神经分化提供一个真实的三维环境。将小鼠胚胎干细胞和诱导多能干细胞植入RADA16支架中，应用于体外帕金森疾病模型，结果显示与层粘连蛋白包被的二维环境和基质胶的三维环境相比，自组装短肽水凝胶支架能提高多巴胺能神经元分化和成熟的基因表达水平，显著增强多巴胺的释放。 2.2.3 治疗脊髓损伤脊髓损伤的病理生理学特点包括创伤后炎症反应、细胞凋亡和脱髓鞘发生，导致组织瘢痕和髓内空腔形成，影响神经修复。星形胶质瘢痕形成和神经再生抑制是脊髓损伤恢复中的两大难题。Guo等[24]分离出许旺细胞和神经祖细胞种植于RADA16自组装短肽水凝胶支架中，再将水凝胶支架移植入脊髓损伤大鼠脊柱中，发现移植细胞迁移，其轴突能够长入纳米纤维支架中，第一次观察到了脊髓修复，证明自组装短肽水凝胶支架在受损脊髓组织中起桥联作用。随后，他们又进行了一项研究来评估纳米纤维支架对已产生大脑损伤的协助修复，结果显示纳米级基质在损伤部位进行了组装及再重建，减轻了损伤部位的炎症反应和胶质瘢痕形成[25]。 Cigognini等[26]将骨髓归巢短肽BMHP1序列通过甘氨酸连接于RADA16自组装短肽支架上，注入大鼠脊髓损伤模型中，结果显示GAP-43、神经营养因子和细胞外基质重塑蛋白基因表达均上调，基底膜增厚，新生的轴突可长入囊肿中，大鼠后肢运动功能和前后肢协调功能明显改善。Tavakol等[27]同样将BMHP1连于RADA16序列中，研究了该水凝胶支架对人子宫内膜基质细胞的影响和慢性脊髓组织损伤的恢复，分析证明了BMHP1纳米纤维支架能诱导人子宫内膜基质细胞向神经元分化，增强轴突再生，减轻炎症反应的同时可改善运动神经元功能。Tavakol等[28-29]又进一步发现在脊髓损伤模型中，适当增加水凝胶支架的硬度可能通过激活Wnt/β-Catenin信号通路的同时抑制了BMP-4信号通路，改善神经再生并减少星形胶质细胞的发生。体内实验显示，水凝胶的硬度对GFAP阳性细胞上调和运动神经元恢复有明显的效用。因此推断存在一个明确的临界浓度，过高或过低的浓度都会通过不同的分子途径改变细胞行为和神经元分化。短肽序列IKVAV通过结合α6整合素位点激活FAK、JNK、PI3/Akt和ERK信号通路，诱导轴突生长[30]，与β1整合素位点结合抑制星形胶质细胞分化[31]。Tavakol等[32]研究发现，较长的层粘连蛋白序列CQAASIKVAV比IKVAV能更好地模拟活性位点构象，将其连于RADA16主链上，较BMHP序列短肽水凝胶支架能显著提高人子宫内膜基质细胞向神经元分化。基因表达结果显示，TH基因和Bcl-2基因过表达及GFAP基因抑制，短肽序列通过β-Catenin信号通路上调Bcl-2基因促进神经、轴突再生，抑制凋亡。 Shi等[21]发现将人脐带间充质干细胞在与活性星形胶质细胞共培养时，细胞的增殖及神经元分化增强。于是将人脐带间充质干细胞与星形胶质细胞共同种植于RADA16-BDNF水凝胶支架中，所得的共培养体系可促进脑源性神经营养因子放入分泌，增加外源人脐带间充质干细胞向神经元的分化及内源性的神经再生。移植细胞的短肽水凝胶支架对中、小型脑损伤空腔有较好的治疗效果。 Gelain等[20]将Ac-FAQ-LDLK12自组装短肽水凝胶支架注入急性脊髓损伤模型中，结果显示其能促进神经组织的再生，改善运动功能。Cigognini等[33]将RADA16-4G-BMHP1和Ac-FAQ-LDLK12水凝胶注入脊髓损伤部位，两种短肽在损伤后第3天都能发挥较好的止血作用，在第28天都能观察到轴突再生，明显改善脊髓血肿。 Liu等[34]将QL6水凝胶注入脊髓损伤大鼠模型脊髓组织中，发现QL6水凝胶可明显减少创伤后的细胞凋亡、炎症反应和星形胶质细胞增生，发挥组织保护作用。此外，QL6还能促进轴突再生。体外实验发现，QL6水凝胶支架可增强神经元分化，抑制星形胶质细胞发育。神经电生理结果显示，QL6水凝胶可明显改善轴突功能，包括传导速度增加、减少不应性、增强高频传导。Iwasaki等[35]将QL6联合神经干细胞共同注射入颈髓损伤大鼠，发现QL6能减少脊髓损伤囊性空洞的体积，促进神经干细胞分化为运动神经元，减轻病灶周围的炎症反应。QL6和神经干细胞的联合作用可改善大鼠前肢的神经行为功能，增加大鼠前肢活动区域及步长。Zweckberger等[36]在脊髓损伤急性期后2周将QL6自组装短肽支架注射至损伤中心，将神经前体细胞注射于临近脊髓，联合一个缓释生长因子的微泵以保证细胞存活率，结果显示，水凝胶基质可有效提高细胞的生存能力和分化潜能，减少瘢痕组织形成。后续又将神经前体细胞种于QL6支架中联合治疗脊髓损伤，发现联合治疗组中神经元和少突胶质细胞增多，脊髓髓内囊肿体积减小，硫酸软骨素蛋白聚糖沉积减少，突触连接增加，皮质脊髓束行为学结果得到改善。结果表明QL6支架可优化脊髓损伤后微环境，并协同加强神经干细胞治疗效果[37]。Zhao等[38]将神经前体细胞和QL6自组装短肽注射于损伤部位尾侧及吻侧，行为学分析显示，自组装短肽和神经前体细胞联合移植组可显著改善运动功能评分，增强存活率。联合移植组同样可改善神经传导速度，但并没有影响空腔体积。Ando等[39]设计的自组装短肽水凝胶SPG-178支架可诱导神经营养因子治疗脊髓损伤，体外实验证明SPG-178可增加神经生长因子、脑源性神经营养因子、NT-4、TrkA和TrkB的表达，促进运动神经元的神经突生长，体内实验显示使用SPG-178能够提高神经营养因子和神经生长因子的表达，减少炎症反应和胶质瘢痕。 2.2.4 治疗创伤性脑损伤现阶段创伤性脑损伤仍是一个临床难题，因直接的脑损伤和后续炎症反应及贫血造成的持续性继发性神经损伤，可引发大脑组织缺陷和严重残疾[40]。Wang等[41]通过调节RADA16-SVVYGLR短肽水凝胶的pH值和离子浓度等微环境因素，使该水凝胶支架表现出多种理化特性。这种水凝胶支架可支持内皮细胞形成管道样结构，促进神经干细胞的存活和增殖。在体内斑马鱼大脑损伤模型实验中，RADA16- SVVYGLR水凝胶移植组出现了向外生长的血管和神经元发育，有效恢复了横断的视顶盖功能。Shi等[21]为了促进移植的人脐带间充质干细胞向损伤脑组织迁移，将人脐带间充质干细胞与趋化因子受体CXCR4联合，发现移植了混有人脐带间充质干细胞、CXCR4和活化星形胶质细胞的水凝胶支架，可修复中等尺寸大小的创伤性脑损伤空腔。Sun等[42]将设计的短肽应用于脑出血、坐骨神经缺损和脊髓横断模型中，发现相比RADA16支架，RADA16-IKVAV和RADA16-RGD水凝胶支架可为神经再生提供一个更为宽松的环境。 2.2.5 治疗外周神经损伤外周神经较脆且易损伤，通常会导致神经组织、运动和感觉功能丧失。损伤修复的关键在于连接受损组织和创造一个允许轴突通过受损组织再生的环境。Wu等[43]将功能化序列IKVAV和RGD修饰于RADA16-1上，再将两种新合成的肽混合得到纳米纤维水凝胶RADA16-Mix，它克服了RADA16-1酸性较强的主要缺点。最近的研究将RADA16-Mix水凝胶移植于大鼠坐骨神经横断间隙中，对轴突的再生效果进行检测，同时与传统的RADA16-1进行比较。结果发现，RADA16-1水凝胶中再生的神经只是沿着水凝胶形成的空腔表面生长，而RADA16-Mix水凝胶中神经则长入水凝胶中并沿远端生长。同时，RADA16-Mix水凝胶诱导了更多的轴突再生和许旺细胞迁移，促进了更好的功能恢复和新的神经肌肉连接结构形成。结果表明，功能化的RADA16-Mix水凝胶可提供一个更好的外周神经再生环境，以促进外周神经损伤修复。Nune等[44]将PLGA与RADA16-Ⅰ-BMHP1混合得到一种新型纳米纤维体，发现其可有效增强许旺细胞的黏附和分化，同时促进神经发育标志基因SEM3F、NRP2和PLX1的表达。因此，自组装短肽水凝胶支架在外周神经功能再生方面有广阔的前景。 "

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