WO2021227255A1 - 一种融合有nt3的丝素蛋白神经移植物的制备方法 - Google Patents

一种融合有nt3的丝素蛋白神经移植物的制备方法 Download PDF

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WO2021227255A1
WO2021227255A1 PCT/CN2020/104048 CN2020104048W WO2021227255A1 WO 2021227255 A1 WO2021227255 A1 WO 2021227255A1 CN 2020104048 W CN2020104048 W CN 2020104048W WO 2021227255 A1 WO2021227255 A1 WO 2021227255A1
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silk fibroin
fused
nerve graft
solution
preparation
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French (fr)
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刘炎
刘梅
顾晓松
管徒晨
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南通大学
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Priority to US17/633,950 priority Critical patent/US20230144643A1/en
Priority to JP2022504619A priority patent/JP7332218B2/ja
Priority to EP20935613.8A priority patent/EP4011411B1/en
Publication of WO2021227255A1 publication Critical patent/WO2021227255A1/zh

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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
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    • A—HUMAN NECESSITIES
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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  • the invention belongs to the field of biomedical materials, and specifically relates to a silk fibroin nerve graft with NT3 activity and capable of promoting nerve recovery, and can be used in the repair and regeneration of peripheral nerves, spinal cord injuries, and brain injuries.
  • biomaterials are used in nerve repair. Excellent biomaterials should have good biocompatibility and support cell adhesion, migration, cell-cell interaction, proliferation and differentiation. At the same time, these materials also require suitable degradation rates, mechanical properties, and limited immune responses. They have a variety of processing options and can change their structure and morphology according to the specific needs of the tissue.
  • protein is a rational choice for tissue engineering applications. Structural proteins such as collagen, elastin, elastin, albumin, and fibrin are used as sutures, tissue scaffolds, hemostatic agents, and drug delivery agents.
  • Nerve injury includes peripheral nerve injury and central nerve injury.
  • the treatment of long-distance peripheral nerve injury and spinal cord injury often requires the application of different types of grafts.
  • Autologous nerves or xenografts have shortcomings due to lack of sources and limited application scope.
  • Silk protein is a common natural biopolymer material and has a long history of application as a suture thread in the human body. In recent years, it has gradually attracted people's attention as a biological material due to some of its characteristics. Compared with other protein-based biomaterials from the same or heterogeneous tissues, silk fibroin has several main advantages: good biocompatibility, excellent mechanical properties, controllable biodegradability, simple processing technology and so on.
  • the silk fibroin polymer exhibits different processing plasticity.
  • a variety of matrix configurations can be realized, including three-dimensional porous foam, nanofiber, hydrogel, tube and film, which can be applied to various tissue repairs.
  • Neurotrophic factor 3 is a protein encoded by the NTF3 gene. It is an important member of the nerve growth factor family. It can nourish neurons in the peripheral and central nervous system. It can not only help the survival of existing neurons, but also promote new growth. The survival and differentiation of neurons and the connection of synapses.
  • tissue engineering scaffolds constructed only with silk fibroin is relatively single, and the effect of promoting nerve tissue recovery is relatively limited.
  • the instability and limited sources of nutritional factors in the body have greatly restricted their use.
  • the present invention combines the neuroprotective factor 3 (NT-3) active peptide with the silk fibroin light chain peptide to form a fusion protein, and the silk fibroin is self-assembled based on the fusion protein.
  • NT-3 neuroprotective factor 3
  • a new type of nerve graft that can provide good mechanical support and can stably exert neuroprotective function for a long time.
  • a preparation method of NT3 fused silk fibroin nerve graft includes the following steps:
  • the fusion protein and the silk fibroin solution are mixed to obtain the mixed protein solution, the silk fibroin fiber net is placed in the mold, and the mixed protein solution is poured into the mold and freeze-dried to form a nerve conduit; after the denaturation treatment, the final product has NT-3 activity Silk fibroin nerve graft. Further, the silk fibroin fiber web is woven from silk fibroin fibers with a knitting machine.
  • the preparation method of the silk fibroin solution is as follows: dissolving the silk fibroin fibers in a lithium thiocyanate solution, putting the dissolved solution into a dialysis bag, and dialyzing for 60-80 hours with tri-distilled water as the dialysate , Get the silk fibroin solution.
  • the preparation method of the silk fibroin fiber is: boil the mulberry silk in a sodium carbonate solution for not less than 20 minutes, take it out and wash it thoroughly with three-distilled water, repeat this step 2 to 4 times, and get the external The silk fibroin fiber of sericin.
  • the concentration of the fusion protein and the silk fibroin solution is 5%-40%; the mass ratio of the fusion protein to the silk fibroin is 1:99-50:50.
  • the deformation treatment is immersion in 60% ethanol for 10-14 hours.
  • the molecular weight cut-off of the dialysis bag is 12-16kDa.
  • the temperature of freeze-drying is -70°C.
  • the concentration of the lithium thiocyanate solution is 9 mol/L.
  • the present invention also provides a silk fibroin nerve graft fused with NT3, which is prepared by the above-mentioned preparation method.
  • the prior art mainly adopts the method of neurotrophic factor adsorption or covalent attachment to the scaffold.
  • the present invention adopts the biologically active fragment of NT-3 and the light chain silk fibroin to be simultaneously expressed in the fusion protein.
  • the fusion protein is based on silk fibroin.
  • the main body is fused with the active ingredients of NT-3, which can be fixed in the silk fibroin duct along with the silk fibroin. It can play the role of neuroprotection and promote nerve regeneration for a long time without changing the composition of silk fibroin.
  • the main material used in the present invention is silk fibroin, supplemented by silk fibroin light chain fused with NT-3 active peptide segment, and no toxic or side-effect substances such as cross-linking agents, surfactants, etc. are added during processing. , So it has good biocompatibility.
  • FIBL-NT3-L- NT3 has the strongest activity.
  • the silk fibroin conduit of the present invention introduces the silk fibroin light chain fused with NT-3 functional peptide before the silk fibroin is crystallized. Therefore, in the process of silk fibroin self-assembly, the NT-3 active polypeptide It will be fixed in the silk fibroin duct along with the silk fibroin light chain, which can provide mechanical support while exerting neuroprotection and promoting nerve regeneration for a long time. At the same time, the NT-3 active peptide in the nerve duct can be adjusted according to the actual situation. The ratio is conducive to the repair of nerve damage.
  • Figure 1 shows the effects of NT3-containing fusion proteins of different designs on the growth of dorsal root ganglion neurons cultured in vitro;
  • Figure 2 shows the effects of NT3-containing fusion proteins of different designs on the growth of dorsal root ganglion neurons cultured in vitro;
  • a preparation method of NT3 fused silk fibroin nerve graft includes the following steps:
  • a preparation method of NT3 fused silk fibroin nerve graft includes the following steps:
  • Fusion protein sequence (including N-terminal His and flexible Linker):
  • the recombinant expression vector obtained in step 1 was transferred into BL21 E. coli, the expression was induced at 37°C and then ultrasonically broken, and the fusion protein was purified and identified using Ni-NTA to obtain the fusion protein.
  • the silk fibroin fiber obtained in step 3 is woven into a silk fibroin fiber web with a braiding machine
  • step 6 Mix the fusion protein obtained in step 2 and the silk fibroin solution obtained in step 4 in a certain proportion to obtain a mixed protein solution.
  • concentration of the mixed protein solution is configured to be 5%-40%, and the quality of the fusion protein and silk fibroin The ratio is 1:99-50:50, put the silk fibroin fiber net obtained in step 5 in a mold, pour the mixed protein solution into the mold and freeze-dry at -70°C.
  • the silk fibroin, fusion protein and silk fibroin fiber The nets are cross-linked to form nerve ducts.
  • the silk fibroin fusion NT-3, FIBL, FIBL-NT3, FIBL-linker-NT3, FIBL-(NT3) 2 promotes the growth of DRG axons ( Figure 1) and statistical map ( Figure 2), the statistical data comes from The average value of the axon length counts of 30 neurons in each group. It can be seen from the figure that compared with the blank group, NT3 can promote the axon growth of cells, and FIBL-NT3, FIBL-linker-NT3, and FIBL-(NT3) can more effectively promote axon growth.

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Abstract

一种融合有NT3的丝素蛋白神经移植物及其制备方法，步骤包括：合成包含有丝素蛋白轻链以及NT‑3的基因片段，连接至pET‑30表达载体，将得到的重组表达载体转入BL21大肠杆菌，得到融合蛋白；将丝素纤维网置于模具中，混合融合蛋白和丝素蛋白溶液，冷冻干燥，丝素蛋白与融合蛋白交联形成神经导管；变形处理后最终获得具有NT‑3活性的丝素蛋白神经移植物。该丝素蛋白神经移植物在提供力学支撑的同时能够长时间的发挥神经保护以及促进神经再生的作用，同时可以根据实际情况调整神经导管中NT‑3活性肽的比例，利于神经损伤的修复。

Description

一种融合有NT3的丝素蛋白神经移植物的制备方法 技术领域
本发明属于生物医学材料领域，具体涉及一种具有NT3活性、能够促进神经恢复的丝素蛋白神经移植物，可在周围神经、脊髓损伤、脑损伤修复再生中应用。
背景技术
随着组织工程、生物材料学等新兴学科的发展，越来越多的生物材料被用在神经修复上。优秀的生物材料应该具有良好的生物相容性，支持细胞的黏附、迁移、细胞间的相互作用、增殖以及分化。同时这些材料还需要合适的降解速率，力学性质以及有限的免疫反应，具有多种加工选择，可以根据组织的具体需要改变结构和形态。蛋白质作为天然组织的组成部分，是组织工程应用的理性选择。结构蛋白如胶原蛋白、弹力蛋白、弹性肽、白蛋白和纤维蛋白被用作缝合线、组织支架、止血剂和药物递送剂。
神经损伤包括周围神经损伤和中枢神经损伤，目前长距离周围神经损伤和脊髓损伤的治疗往往需要应用不同类型的移植物，自体神经或者异种移植物等由于来源匮乏，应用范围受限等缺点使得人们开始寻找新的组织工程移植物替代物上。蚕丝蛋白是一种常见的天然生物高分子材料，作为缝合线在人体中有着悠久的应用历史。近年来，由于它的一些特性作为生物材料逐渐受到了人们的关注。与来自同种或异种来源组织的其他蛋白基生物材料相比，丝素蛋白有几个主要优势：良好的生物相容性，优异的机械性能，可控生物降解性，加工工艺简单等等。同时丝素蛋白聚合物表现出不同的加工可塑性，通过制造技术的变化，可以实现多种基质配置，包括三维多孔泡沫、纳米纤维、水凝胶、管和薄膜，可以应用多种组织修复。神经营养因子3是由NTF3基因编码的一种蛋白，是神经生长因子家族的重要成员，可以营养周围和中枢神经系统的神经元，不仅能够有助于现有神经元的存活，还能促进新生神经元的存活和分化以及突触的连接。
目前仅仅使用丝素蛋白构建的组织工程支架功能较为单一，且促进神经组 织的恢复作用较为有限。有研究通过将神经移植物浸泡在营养因子(如NGF)中或与丝素蛋白混合后冻干形成导管，在前期具有良好的促神经生长作用，但这种作用会随着时间的延长逐渐衰弱，同时营养因子在体内的不稳定性以及有限的来源使得它们的使用受到了很大的限制。
发明内容
本发明针对现有技术的不足，将神经保护因子3(NT-3)活性肽段与丝素蛋白轻链肽段相结合构成融合蛋白，以此融合蛋白为基础进行丝素蛋白的自组装获得了一种既能提供良好的力学支撑又能长时间稳定发挥神经保护功能的新型神经移植物。
本发明的技术方案如下：
一种融合有NT3的丝素蛋白神经移植物的制备方法，包括以下步骤：
合成包含有丝素蛋白轻链以及NT-3的基因片段，连接至pET-30表达载体，将得到的重组表达载体转入BL21大肠杆菌，得到融合蛋白；
将融合蛋白以及丝素蛋白溶液混合，得到混合蛋白溶液，将丝素纤维网置于模具中，将混合蛋白溶液倒入模具中冷冻干燥，形成神经导管；变性处理后最终获得具有NT-3活性的丝素蛋白神经移植物。进一步的，所述丝素纤维网由丝素纤维用编织机编织而成。
进一步的，所述丝素蛋白溶液的制备方法为：将丝素纤维放入硫氰酸锂溶液中进行溶解，溶解后的溶液装入透析袋，以三蒸水为透析液透析60～80小时，得到丝素蛋白溶液。
进一步的，所述丝素纤维的制备方法为：将桑蚕丝置于碳酸钠溶液中煮沸不少于20分钟，取出后用三蒸水充分洗涤，重复此步骤2～4次，得到脱去外部丝胶的丝素纤维。
进一步的，融合蛋白和丝素蛋白溶液的浓度为5％-40％；融合蛋白与丝素蛋白的质量比例为1:99～50:50。
进一步的，变形处理为浸泡在60％乙醇中处理10～14h。
进一步的，透析袋的截留分子量为12～16kDa。
进一步的，冷冻干燥的温度为-70℃。
进一步的，硫氰酸锂溶液的浓度为9mol/L。
本发明还提供一种融合有NT3的丝素蛋白神经移植物，由上述制备方法制备而成。
有益效果
现有技术主要采用神经营养因子吸附或共价连接结合到支架的方法，本发明采用NT-3的生物活性片段和轻链丝素蛋白同时表达在融合蛋白，该融合蛋白是以丝素蛋白为主体融合NT-3的有效成分，能够随着丝素蛋白一同被固定在丝素导管中。能够在不改变丝素蛋白的成分的前提下，能够长时间的发挥神经保护以及促进神经再生的作用。
本发明中所用到的主要材料为丝素蛋白，辅以融合NT-3活性肽段的丝素蛋白轻链，在加工过程中没有加入例如交联剂、表面活性剂等任何有毒、副作用的物质，因此具有良好的生物相容性。
本发明所述的丝素融合蛋白在体外与神经组织细胞进行共培养时，通过形态学观察，NT-3相关因子的表达测定，均显示出明显的促生长作用，其中FIBL-NT3-L-NT3的活性最强。
本发明中所述的丝素导管，在丝素蛋白结晶化之前引入了融合有NT-3功能肽段的丝素蛋白轻链，因此在丝素蛋白自组装的过程中，NT-3活性多肽将随着丝素轻链一同被固定在丝素导管中，在提供力学支撑的同时能够长时间的发挥神经保护以及促进神经再生的作用，同时可以根据实际情况调整神经导管中NT-3活性肽的比例，利于神经损伤的修复。
附图说明
图1为不同设计的含NT3的融合蛋白对体外培养的背根节神经元突起生长的影响；
图2为不同设计的含NT3的融合蛋白对体外培养的背根节神经元突起生长的影响；
具体实施方式
实施例1
一种融合有NT3的丝素蛋白神经移植物的制备方法，包括以下步骤：
1.丝素融合蛋白的表达及纯化：构建重组表达载体，表达目的蛋白后进行纯化；
2.蚕丝丝素纤维的获取：取桑蚕丝生丝，去除丝胶，获得桑蚕丝丝素纤维；
3.丝素蛋白溶液的制备；
4.丝素纤维网的制备；
5.将丝素纤维网放置在模具当中，将步骤1和3获得的蛋白溶液混合后导入模具当中，冷冻干燥使丝素蛋白进行自组装后形成神经导管。
实施例2
一种融合有NT3的丝素蛋白神经移植物的制备方法，包括以下步骤：
1.重组表达载体的构建：合成包含有丝素蛋白轻链以及NT-3的基因片段，连接至pET-30表达载体，完成重组表达载体的构建，含有标签tag和linker的不同融合蛋白序列如下所示。
融合蛋白序列(包含N端His和柔性Linker)：
FIBL Protein Length＝253 MW＝26898.8 pI＝6.10
Figure PCTCN2020104048-appb-000001
FIBL-NT3 Protein Length＝372 MW＝40503.7 pI＝7.39
Figure PCTCN2020104048-appb-000002
FIBL-linker-NT3 Protein Length＝382 MW＝41134.1 pI＝7.39
Figure PCTCN2020104048-appb-000003
FIBL-(NT3)2 Protein Length＝501 MW＝54739.0 pI＝8.28
Figure PCTCN2020104048-appb-000004
2.融合蛋白的表达及纯化：将步骤1获得的重组表达载体转入BL21大肠杆菌，37℃诱导表达后超声破碎，使用Ni-NTA纯化融合蛋白并鉴定，得到融合蛋白。
3.取适量的桑蚕丝，置于0.2％的碳酸钠溶液中煮沸30分钟，取出后用三蒸水充分洗涤，重复此步骤三次，得到脱去外部丝胶的丝素纤维，置于超净台晾干备用。
4.将丝素纤维放入9M的硫氰酸锂溶液中进行溶解，溶解后的溶液装入透析袋(截留分子量约为14kDa)，以三蒸水为透析液透析72小时，得到丝素蛋白 溶液。
5.将步骤3获得的丝素纤维用编织机编织丝素纤维网；
6.将步骤2得到的融合蛋白以及步骤4得到的丝素蛋白溶液按照一定比例混合，得到混合蛋白溶液，将混合蛋白溶液的浓度配置为5％-40％，融合蛋白与丝素蛋白的质量比例为1:99～50:50，将步骤5获得的丝素纤维网置于模具中，将混合蛋白溶液倒入模具中置于-70℃冷冻干燥，丝素蛋白、融合蛋白和丝素纤维网交联形成神经导管。
7.浸泡在60％乙醇中进行变形处理12h，三蒸水洗涤后晾干，最终获得了具有NT-3活性的丝素蛋白神经移植物。
融合NT-3，FIBL，FIBL-NT3,FIBL-linker-NT3，FIBL-(NT3) 2的丝素蛋白促进DRG轴突的生长图(图1)和统计图(图2)，统计图数据来自各组30个神经元轴突长度计数的平均值。从图中可以看出，相对空白组,NT3能够促进细胞的轴突生长，FIBL-NT3,FIBL-linker-NT3，FIBL-(NT3)能够更加有效促进轴突生长。

Claims (10)

  1. 一种融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，包括以下步骤：
    合成包含有丝素蛋白轻链以及NT-3的基因片段，连接至pET-30表达载体，将得到的重组表达载体转入BL21大肠杆菌，得到融合蛋白；
    将融合蛋白以及丝素蛋白溶液混合，得到混合蛋白溶液，将丝素纤维网置于模具中，将混合蛋白溶液倒入模具中冷冻干燥，形成神经导管；变形处理后最终获得具有NT-3活性的丝素蛋白神经移植物。
  2. 根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，所述丝素纤维网由丝素纤维用编织机编织而成。
  3. 根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，所述丝素蛋白溶液的制备方法为：将丝素纤维放入硫氰酸锂溶液中进行溶解，溶解后的溶液装入透析袋，以三蒸水为透析液透析60～80小时，得到丝素蛋白溶液。
  4. 根据权利要求2或3所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，所述丝素纤维的制备方法为：将桑蚕丝置于碳酸钠溶液中煮沸不少于20分钟，取出后用三蒸水充分洗涤，重复此步骤2～4次，得到脱去外部丝胶的丝素纤维。
  5. 根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，混合蛋白溶液的浓度为5％-40％；融合蛋白与丝素蛋白的质量比例为1:99～50:50。
  6. 根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，变形处理为浸泡在60％乙醇中处理10～14h。
  7. 根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，透析袋的截留分子量为12～16kDa。
  8. 根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，冷冻干燥的温度为-70℃。
  9. 根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法，其特征在于，硫氰酸锂溶液的浓度为9mol/L。
  10. 一种融合有NT3的丝素蛋白神经移植物，其特征在于，由权利要求1～9 任一项所述的制备方法制备而成。
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