CN114481356B - 一种仿蚕茧支架及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种仿蚕茧支架及其制备方法和应用。本发明的仿蚕茧支架，具有独特的核壳结构，可以针对化学和物理特性进行优化，能够以最小的低温损伤对生物样品进行低温保存。它不仅可以促进细胞的粘附和生长，还可以实现冻存细胞的快速均匀复温，并且在冻融后最大限度地维持了细胞的活力和增殖，从而能够有效冻存免疫细胞和干细胞。干细胞‑仿蚕茧支架构建的组织工程支架在冻存后保留了较高的细胞存活率，可以直接植入受损部位，促进组织再生和修复。因此，仿蚕茧支架在细胞和组织工程支架的长期保存方面具有较高的安全性和有效性，在基础研究、细胞治疗、组织工程等领域具有广阔的应用前景。
Description
技术领域
本发明涉及表面修饰、细胞冻存、组织修复及疾病治疗等材料制备和生物医学领域，尤其涉及集静电纺丝、原位表面功能化和冷冻成形于一体的自下而上的新型制备技术，获得一种仿蚕茧支架。
背景技术
细胞治疗包括将健康细胞引入患者体内以缓解棘手的身体状况，已逐渐成为传统治疗的有效替代方法，为人类提供了新的治疗选择。然而，目前仍然存在一些障碍阻碍了细胞治疗在临床上的广泛应用，例如与胚胎干细胞相关的伦理争议、治疗欺诈案例以及患者的细胞生物安全性。尤其是，细胞是具有活性的生物物种，难以获得足够的高质量的细胞用于患者移植，这极大地限制了其应用范围。为了满足细胞治疗的需求，迫切需要开发一种有效的方法来改善细胞和组织工程的生产、储存和运输，直接用于临床实践。近年来，细胞冷冻保存已发展成为一项重要的技术，可在极低的温度(-80℃或-196℃)下保存细胞，不仅能维持细胞活力，还能最大限度地减少细胞中的基因改变、转化和老化。然而，传统的低温保存方法，如缓慢冷冻和玻璃化冷冻，存在细胞毒性冷冻保护剂、渗透性休克和冷冻损伤等问题，导致细胞复苏后质量差。因此，一种可靠的冻存方法对于支持细胞治疗是极其关键的。
目前，众多基于不同机制的先进生物技术已应用于细胞冻存，包括采用海藻糖、三维(3D)支架和纳米技术，显著提高了解冻后细胞的存活率。特别是3D支架，它具有独特的细胞外基质样结构，为细胞提供了最佳的生长环境，也为细胞的高效冻存提供了强有力的工具。研究表明，3D支架能够在冻融后维持细胞的原始形态、生物学功能，甚至细胞间的相互作用，保护细胞免受冻-融过程中的机械损伤。然而，由天然或合成聚合物组成的3D支架，面临导热性差和力学性能差的问题，这降低了细胞冻存效果。此外，传统3D支架的抗冰晶能力较差，且冷冻保存后会发生机械或结构损伤，导致细胞存活率低。最重要的是，大多数支架材料由于生物相容性差，不能很好地将细胞保存与细胞治疗相结合，极大地限制了复苏细胞在临床上的直接应用。因此，设计和制造具有适当物理几何形状和生物化学性质的3D支架成为低温保存的主要目标。
蚕茧以其独特的结构、组分和性能被认为是生物工程结构复合材料的理想天然原型。蚕茧具有高度优化的层次结构，具有良好的刚度和强度，可以保护蚕蛹免受各种外在环境威胁。从宏观上看，蚕丝纤维是一种天然的蛋白质-聚合物复合物，内有丝素蛋白，外有粘胶状丝胶蛋白。这种核壳结构使蚕茧有利于为蚕蛹提供适宜的生长温度，维持蚕蛹在恶劣天气条件下的代谢活动。丝素蛋白和丝胶蛋白作为蚕茧的主要结构成分，不仅具有良好的生物相容性，能够促进细胞和组织的附着和生长，同时还具有抗氧化、抗凝血、促进伤口愈合和防护紫外线等特性，被食品和药物管理局批准用于医疗器械。重要的是，丝胶蛋白可以有效抑制冰晶生长，在细胞冻存中可提高细胞的冻存效率。
本专利，我们采用静电纺丝、原位表面功能化和冷冻成形相结合的自底向上制备技术，开发了一种仿蚕茧支架，用于细胞冻存和组织工程领域。
发明内容
本发明的目的在于提供一种仿蚕茧支架。其特征在于仿蚕茧的细胞冻存支架具有核-壳结构，由丝素蛋白、丝胶蛋白、聚丙氨酸通过静电纺丝、原位表面功能化和冷冻成形相结合的自底向上制备技术制备而成。为此，本发明的技术方案为：
1)将丝素蛋白、聚丙氨酸以及聚氧化乙烯水溶液进行混合，搅拌均匀，得到电纺溶液；
2)采用静电纺丝装置，将步骤1)制备得到的电纺溶液装入微量注射泵中，调节电纺溶液的流量、静电纺的电压以及接收平板与注射泵针尖的距离，使丝素蛋白电纺溶液在电场力的作用下形成稳定的射流，采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基琥珀酰亚胺溶液作为接收池，收集纤维。
3)将步骤2)收集纤维加入丝胶蛋白溶液中，室温下继续反应，除去反应液，蒸馏水洗涤3次，冷冻干燥，收集纤维支架。
步骤1)的具体操作为：
1a)制备丝素蛋白、聚丙氨酸水溶液；
1b)制备聚氧化乙烯水溶液；
1c)将聚氧化乙烯加入1a)制备的溶液中，继续搅拌均匀，得到电纺溶液。
步骤1a)的具体操作为：将家蚕的茧切成小块，用去离子水配好的0.02M碳酸钠溶液煮沸。煮沸1小时，分离上清和未溶物，未溶物水洗，3次，20分钟/次。晾干后每按1∶50(g/mL)放入溴化锂溶液(9.3M)中，60℃放置4小时。溶解后，溶液变成透明琥珀色。透析(Mw12000)2天，9000rpm，离心20分钟，弃沉淀，冷冻干燥3天，获得丝素蛋白。配成丝素蛋白母液(5-20％w/w)。
步骤1a)中聚丙氨酸的浓度为丝素蛋白质量的1-20％。
步骤1c)中聚氧化乙烯的浓度为丝素蛋白质量的0.01-1％。
步骤2)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基琥珀酰亚胺溶液质量浓度分别为30-50％、15-25％。
步骤3)中室温反应的时间为0.5-72小时，丝胶蛋白的质量浓度为0.5-10％。
本发明具有以下有益效果：利用本发明的具有蚕茧仿生特性和功能的仿蚕茧支架，可以进行细胞的体外扩增、提高细胞冻存效率，为细胞治疗、组织工程提供稳定高质量的细胞来源。
本发明的仿蚕茧支架具有以下优点：
(1)采用的材料生物相容性好，制备简单，无细胞毒性。
(2)可以实现细胞的体外培养和扩增。
(3)提高细胞冻存效率。
(4)为组织工程提供稳定的、高质量的细胞来源。
本发明的仿蚕茧支架，具有独特的核壳结构，可以针对化学和物理特性进行优化，能够以最小的低温损伤对生物样品进行低温保存。本发明的仿蚕茧支架不仅可以促进细胞的粘附和生长，还可以实现冻存细胞的快速均匀复温，并且在冻融后最大限度地维持了细胞的活力和增殖，从而能够有效冻存免疫细胞和干细胞。干细胞-仿蚕茧支架构建的组织工程支架在冻存后也具有较高的细胞存活率，可以直接植入伤口，促进组织再生。因此，本发明的仿蚕茧支架在细胞和组织工程支架的长期保存方面具有较高的安全性和有效性，在基础研究、细胞治疗、组织工程等领域具有广阔的应用前景。
附图说明
图1为本发明实施例1制备的仿蚕茧支架实体图。
图2为本发明实施例1制备的仿蚕茧支架扫描电镜图。
图3为本发明实施例1制备的仿蚕茧支架中性粒细胞冻存2天后死活染色。
具体实施方式
通过下述实施例将有助于进一步理解本发明，但本发明并不受其限制。
实施例1
(1)将家蚕的茧切成小块，用去离子水配好的0.02M碳酸钠溶液煮沸。煮沸1小时，分离上清和未溶物，未溶物水洗，3次，20分钟/次。晾干后每按1∶50(g/mL)放入溴化锂溶液(9.3M)中，60℃放置4小时。溶解后，溶液变成透明琥珀色。透析(Mw 12000)2天，9000rpm，离心20分钟，弃沉淀，冷冻干燥3天，获得丝素蛋白。
(2)将步骤(1)中上清在水中透析(Mw 12000)2天，除去碳酸钠溶液，冻干收集，得到丝胶蛋白。
(3)将步骤(1)中制备的丝素蛋白溶于六氟异丙醇中制成7.5％w/v的溶液
(4)将5％w/w聚丙氨酸添加到步骤(3)制备的溶液中并搅拌4小时。
(5)将聚氧化乙烯(0.1％w/w)添加到步骤(4)制备的混合溶液中，继续搅拌4小时。
(6)将步骤(5)制备的溶液转移到注射器中进行静电纺丝。通过针头向溶液施加20kV正电压，流速固定在0.5mL/h。采用30％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和15％的N-羟基磺基琥珀酰亚胺(EDC/NHS)溶液收集纤维。
(7)将步骤(6)制备的纤维加入5％丝胶蛋白溶液中，室温下反应24小时，取出纤维，用蒸馏水洗涤3次，冷冻干燥，获得仿蚕茧支架(图1)。
利用扫描电镜观察了仿蚕茧支架的形貌，如图2所示，仿蚕茧支架具有连续的丝状结构。
实施例2
将中性粒细胞与实施例1制备的仿蚕茧支架共培养30分钟，用1mL细胞冷冻液代替培养基并转入冻存管中。然后，将冻存管放入冷冻盒(Nalgene，在-80℃冰箱中的冷冻速率为1℃/min)过夜，然后在液氮中储存2天。37℃水浴复苏后，消化收集细胞，采用活死细胞染色试剂盒对细胞进行染色。实验结果活细胞比例为69％(图3)。
实施例3
将人脐带血间充质干细胞与实施例2制备的仿蚕茧支架共培养30分钟，用1mL细胞冷冻液代替培养基并转入冻存管中。然后，将冻存管放入冷冻盒(Nalgene，在-80℃冰箱中的冷冻速率为1℃/min)过夜，然后在液氮中储存2天。37℃水浴复苏后，消化收集细胞，采用活死细胞染色试剂盒对细胞进行染色。实验结果活细胞比例为90.9％。
应当说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应该理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (6)
1.一种仿蚕茧支架的制备方法，其特征在于仿蚕茧支架具有核-壳结构，由丝素蛋白、丝胶蛋白、聚丙氨酸、聚氧化乙烯通过静电纺丝、原位表面功能化和冷冻成形相结合的自底向上制备技术制备而成，具体包括以下步骤：
1)将丝素蛋白、聚丙氨酸以及聚氧化乙烯水溶液进行混合，搅拌均匀，得到电纺溶液；
2)采用静电纺丝装置，将步骤1)制备得到的丝素蛋白电纺溶液装入微量注射泵中，调节电纺溶液的流量、静电纺的电压以及接收平板与注射泵针尖的距离，使电纺溶液在电场力的作用下形成稳定的射流，采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基琥珀酰亚胺溶液作为接收池，收集纤维；
3)将步骤2)收集的纤维加入丝胶蛋白溶液中，室温下继续反应，除去反应液，蒸馏水洗涤，冷冻干燥，收集纤维支架。
2.根据权利要求1所述的一种仿蚕茧支架的制备方法，其特征在于：步骤1)中丝素蛋白的质量浓度为5-20％，聚丙氨酸的浓度为丝素蛋白的1-20％，聚氧化乙烯的浓度为丝素蛋白质量的0.01-1％。
3.根据权利要求1所述的一种仿蚕茧支架的制备方法，其特征在于：步骤2)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基琥珀酰亚胺溶液质量浓度分别为30-50％、15-25％。
4.根据权利要求1所述的一种仿蚕茧支架的制备方法，其特征在于：步骤3)中室温反应的时间为0.5-72小时，丝胶蛋白的质量浓度为0.5-10％。
5.权利要求1所述方法制备得到的仿蚕茧支架在细胞冻存、组织工程领域的应用。
6.根据权利要求5所述的应用，其特征在于所述的细胞为肿瘤细胞、免疫细胞、干细胞。
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