BP Bioprocess 2164-5566 Scientific Research Publishing 10.12677/BP.2021.114014 BP-46647 BP20210400000_98250409.pdf 生命科学 精氨酸–谷氨酸–天冬氨酸–缬氨酸改性丝素导管的制备及性能研究 Study on Preparation and Performance of Arg-Glu-Asp-Val Modified Silk Fibroin Tube 李 文慧 2 1 姚 珂 2 1 张 鲁中 2 1 杨 宇民 2 1 南通大学，教育部/江苏省神经再生重点实验室/神经再生协同创新中心，江苏 南通 null 20 10 2021 11 04 123 133 © Copyright 2014 by authors and Scientific Research Publishing Inc. 2014 This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

目的：制备由REDV多肽修饰且具沟槽结构的丝素蛋白支架。方法：在含有30微米沟槽结构的PDMS膜上制备丝素薄膜，卷成管。通过多巴胺改性在丝素移植物表面共价连接REDV多肽进行修饰。扫描电镜观察其内表面微观形貌，电子万能试验机(TFW-58)检测REDV多肽修饰的丝素蛋白支架力学性能的改变。将RSC96细胞种植在丝素蛋白支架内表面1-3 d，观察丝素蛋白质支架内表面的沟槽结构对细胞趋向生长的影响。使用CCK-8 (cell counting Kit-8)测定法检测RSC96细胞在丝素移植物表面的增殖情况。采用SD大鼠制备坐骨神经损伤模型，分别用丝素导管(SF)、具沟槽丝素导管(TOPOLOGY)、REDV多肽修饰丝素导管(REDV)、REDV修饰且具沟槽结构丝素导管(TOPOLOGY/REDV)进行桥接修复。术后12 w测量靶肌湿重比、移植段坐骨神经纵切免疫荧光染色。结果：扫描电镜观察，TOPOLOGY组及TOPOLOGY/REDV组均具有均一的沟槽结构；力学测试结果表明REDV多肽修饰的丝素蛋白支架力学性能有所改善；RSC96细胞与丝素移植物共培养发现移植物表面沟槽结构对于细胞的趋向生长具有引导作用；CCK-8细胞活力检测表明REDV多肽修饰的丝素蛋白支架较单纯的丝素蛋白支架而言生物相容性有所改善；术后12 w靶肌恢复湿重比及坐骨神经纵切免疫荧光染色结果表明REDV多肽修饰且具沟槽结构的丝素蛋白支架对于细胞的吸附生长、损伤神经的修复具有促进作用。结论：REDV多肽修饰且具沟槽结构的丝素蛋白支架具有良好的生物相容性，对于细胞的粘附及趋向生长具有促进作用，为丝素蛋白在神经损伤再生领域提供了新的应用。 Purpose: To prepare a silk fibroin scaffold with grooved structure modified by REDV polypeptide. Method: A silk fibroin film was prepared on a PDMS film containing a 30-micron groove structure and rolled into a tube. The REDV polypeptide is covalently attached to the surface of the silk fibroin graft for modification by dopamine modification. Scanning electron microscope was used to observe the micro morphology of the inner surface, and the electronic universal test (TFW-58) machine was used to detect the changes in the mechanical properties of the silk fibroin scaffold modified with REDV polypeptide. RSC96 cells were planted on the inner surface of the silk fibroin scaffold for 1 - 3 days, and the influence of the groove structure on the inner surface of the silk fibroin scaffold on cell growth was observed. CCK-8 (cell counting Kit-8) assay was used to detect the proliferation of RSC96 cells on the surface of silk fibroin grafts. SD rats were used to prepare sciatic nerve injury models. Silk fibroin catheters (SF), grooved silk fibroin catheters (TOPOLOGY), REDV polypeptide modified silk fibroin catheters (REDV), REDV modified silk fibroin catheters with grooved structure (TOPOLOGY/REDV) were used to perform bridge repair. 12 weeks after the operation, the target muscle wet weight ratio and the longitudinal section of the transplanted sciatic nerve were measured by immunofluorescence staining. Results: Scanning electron microscopy showed that both the TOPOLOGY group and the TOPOLOGY/REDV group had a uniform groove structure; the mechanical test results showed that the mechanical properties of the silk fibroin scaffold modified with REDV polypeptides had been improved; RSC96 cells and silk fibroin grafts were co-cultured, and it was found that the groove structure on the surface of the grafts had a guiding effect on the growth of cells; CCK-8 cell viability test showed that the silk fibroin scaffold modified by REDV polypeptide is more biological than simple silk fibroin scaffold. The CCK-8 cell viability test showed that the silk fibroin scaffold modified by REDV polypeptide has improved biocompatibility compared with the pure silk fibroin scaffold; the target muscle restored wet weight ratio and sciatic nerve longitudinal section immunofluorescence staining results 12 weeks after operation, which shows that the silk fibroin scaffold modifies with REDV polypeptide and has a grooved structure that can promote the adhesion and growth of cells and the repair of damaged nerves. Conclusion: REDV polypeptide modified silk fibroin scaffold with groove structure has good biocompatibility, promotes cell adhesion and growth tendency, and provides a new application for silk fibroin in the field of nerve injury and regeneration.

 丝素导管，多巴胺改性，多肽修饰，神经再生, Silk Fibiroin Dopamine Modification Peptide Modified Nerve Regeneration 摘要

目的：制备由REDV多肽修饰且具沟槽结构的丝素蛋白支架。方法：在含有30微米沟槽结构的PDMS膜上制备丝素薄膜，卷成管。通过多巴胺改性在丝素移植物表面共价连接REDV多肽进行修饰。扫描电镜观察其内表面微观形貌，电子万能试验机(TFW-58)检测REDV多肽修饰的丝素蛋白支架力学性能的改变。将RSC96细胞种植在丝素蛋白支架内表面1-3 d，观察丝素蛋白质支架内表面的沟槽结构对细胞趋向生长的影响。使用CCK-8 (cell counting Kit-8)测定法检测RSC96细胞在丝素移植物表面的增殖情况。采用SD大鼠制备坐骨神经损伤模型，分别用丝素导管(SF)、具沟槽丝素导管(TOPOLOGY)、REDV多肽修饰丝素导管(REDV)、REDV修饰且具沟槽结构丝素导管(TOPOLOGY/REDV)进行桥接修复。术后12 w测量靶肌湿重比、移植段坐骨神经纵切免疫荧光染色。结果：扫描电镜观察，TOPOLOGY组及TOPOLOGY/REDV组均具有均一的沟槽结构；力学测试结果表明REDV多肽修饰的丝素蛋白支架力学性能有所改善；RSC96细胞与丝素移植物共培养发现移植物表面沟槽结构对于细胞的趋向生长具有引导作用；CCK-8细胞活力检测表明REDV多肽修饰的丝素蛋白支架较单纯的丝素蛋白支架而言生物相容性有所改善；术后12 w靶肌恢复湿重比及坐骨神经纵切免疫荧光染色结果表明REDV多肽修饰且具沟槽结构的丝素蛋白支架对于细胞的吸附生长、损伤神经的修复具有促进作用。结论：REDV多肽修饰且具沟槽结构的丝素蛋白支架具有良好的生物相容性，对于细胞的粘附及趋向生长具有促进作用，为丝素蛋白在神经损伤再生领域提供了新的应用。

 关键词

丝素导管，多巴胺改性，多肽修饰，神经再生

 Study on Preparation and Performance of Arg-Glu-Asp-Val Modified Silk Fibroin Tube<sup> </sup>

Wenhui Li, Ke Yao, Luzhong Zhang*, Yumin Yang*

Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Nantong University, Nantong Jiangsu

Received: Oct. 8th, 2021; accepted: Nov. 16th, 2021; published: Nov. 23rd, 2021

 ABSTRACT

Purpose: To prepare a silk fibroin scaffold with grooved structure modified by REDV polypeptide. Method: A silk fibroin film was prepared on a PDMS film containing a 30-micron groove structure and rolled into a tube. The REDV polypeptide is covalently attached to the surface of the silk fibroin graft for modification by dopamine modification. Scanning electron microscope was used to observe the micro morphology of the inner surface, and the electronic universal test (TFW-58) machine was used to detect the changes in the mechanical properties of the silk fibroin scaffold modified with REDV polypeptide. RSC96 cells were planted on the inner surface of the silk fibroin scaffold for 1 - 3 days, and the influence of the groove structure on the inner surface of the silk fibroin scaffold on cell growth was observed. CCK-8 (cell counting Kit-8) assay was used to detect the proliferation of RSC96 cells on the surface of silk fibroin grafts. SD rats were used to prepare sciatic nerve injury models. Silk fibroin catheters (SF), grooved silk fibroin catheters (TOPOLOGY), REDV polypeptide modified silk fibroin catheters (REDV), REDV modified silk fibroin catheters with grooved structure (TOPOLOGY/REDV) were used to perform bridge repair. 12 weeks after the operation, the target muscle wet weight ratio and the longitudinal section of the transplanted sciatic nerve were measured by immunofluorescence staining. Results: Scanning electron microscopy showed that both the TOPOLOGY group and the TOPOLOGY/REDV group had a uniform groove structure; the mechanical test results showed that the mechanical properties of the silk fibroin scaffold modified with REDV polypeptides had been improved; RSC96 cells and silk fibroin grafts were co-cultured, and it was found that the groove structure on the surface of the grafts had a guiding effect on the growth of cells; CCK-8 cell viability test showed that the silk fibroin scaffold modified by REDV polypeptide is more biological than simple silk fibroin scaffold. The CCK-8 cell viability test showed that the silk fibroin scaffold modified by REDV polypeptide has improved biocompatibility compared with the pure silk fibroin scaffold; the target muscle restored wet weight ratio and sciatic nerve longitudinal section immunofluorescence staining results 12 weeks after operation, which shows that the silk fibroin scaffold modifies with REDV polypeptide and has a grooved structure that can promote the adhesion and growth of cells and the repair of damaged nerves. Conclusion: REDV polypeptide modified silk fibroin scaffold with groove structure has good biocompatibility, promotes cell adhesion and growth tendency, and provides a new application for silk fibroin in the field of nerve injury and regeneration.

Keywords:Silk Fibiroin, Dopamine Modification, Peptide Modified, Nerve Regeneration

Copyright © 2021 by author(s) and beplay安卓登录

This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).

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 1. 引言

周围神经缺损是临床上常见的损伤，缺损较短时可通过手术缝合进行临床治疗，当神经缺损长度较大时，无法通过手术缝合缺损神经，必须通过适当的神经移植物来桥接神经损伤间隙和恢复损伤神经功能 [ 1 ] [ 2 ]。神经移植物是一种使用自体组织或者天然、人工材料通过一定的工艺手段加工合成的帮助缺损神经两端连接愈合的通道，具有一定的空间结构及物理性能，对于损伤的神经提供营养支持，促进其神经再生 [ 3 ]。自体神经移植法是神经损伤修复的“金标准”，然而自体供体神经来源有限，且会造成供体神经损伤，对于供体区的手术可能会造成供体部位感染，而异体神经移植存在匹配困难。当神经损伤缺口较长时，人工神经移植物可作为一种替代物 [ 4 ] [ 5 ]。具有取向结构的神经导管对于神经轴突的生长具有引导作用，避免神经错位，有利于受损神经断端的吻合 [ 6 ] [ 7 ]。

生物相容性是生物医学材料重要的指标，人工神经移植物必须具有良好的生物相容性。其中，由于其具有优越的生物相容性、低免疫原性及炎症反应性、可调控的生物降解性，基于丝素蛋白的生物材料被广泛应用于组织工程与缓释体系 [ 8 ] [ 9 ]。但是，丝素蛋白的活性物质改性较为困难。多巴胺改性被认为是一种简单温和的材料改性方法，能够改善材料表面，使得细胞更易于粘附 [ 10 ] [ 11 ]。在弱碱性溶液中，多巴胺可在材料表面自氧化形成聚多巴胺，聚多巴胺为材料表面的二次修饰和功能化提供了便捷途径 [ 12 ]。现有研究表明，其结构中含有的氨基和邻苯二酚官能团能与材料表面的氨基、巯基、亚氨基等官能团发生席夫碱反应或迈克尔加成反应，从而在材料表面形成共价键或非共价键作用 [ 13 ]，还有研究表明，多巴胺改性材料表面容易吸附血清蛋白，从而提高材料的细胞的黏附能力 [ 14 ]。

在支架表面添加细胞外基质粘附生物分子的仿生涂层是恢复健康内皮细胞的一种策略。血管内皮细胞纤维连接蛋白CS5区域包含Arg-Glu-Asp-Val (REDV)肽序列，该序列支持内皮细胞在材料表面的附着和扩散 [ 15 ] [ 16 ]。本文拟采用多巴胺改性方法处理丝素移植物，并在其表面共价连接REDV多肽，以此改善丝素导管的细胞黏附性。材料内表面的均一沟槽结构诱导神经细胞的定向生长，从而促进损伤神经再生。

 2. 实验部分 2.1. 再生丝素的制备 2.1.1. 丝素脱丝胶

将50 g蚕丝(购自南通仙基达茧丝制品有限公司)，置于2000 ml 5% (wt/vol)碳酸钠溶液中煮30 min，煮三次，将脱丝胶蚕丝置于通风橱风干。

 2.1.2. 丝素的溶解、透析、冻干

将脱丝胶的丝素用三元溶液(无水氯化钙：无水乙醇：三蒸水 = 1:1.297:1.068) 80℃水浴溶解，搅拌40 min，装入透析袋(分子量截止12,000∼14,000 Da)，透析三天。透析好的丝素溶液于冷冻干燥机中冻干。

 2.2. 丝素神经导管的制备 2.2.1. 丝素薄膜的制备

0.20 g冻干丝素蛋白溶解在1 mL六氟异丙醇。加入50%经过100目筛网过滤所得的NaCl颗粒，摇匀，倒于含有30微米沟槽结构的PDMS膜上，风干制成丝素膜，再浸泡于无水乙醇中5 h变性处理。

 2.2.2. 丝素导管的制备

将变性的丝素薄膜浸泡于三蒸水中，去除氯化钠。用直径为2 mm的内芯作为模具将丝素薄膜卷成管，并使用丝素蛋白/六氟异丙醇溶液将封口处粘连，60℃烘干并置于无水乙醇中浸泡2 h。

 2.2.3. 多巴胺改性连接REDV多肽

将制备好的丝素导管浸泡在新制的多巴胺溶液(Tris缓冲液，pH = 8.5) 24 h，再用水反复清洗，直至水无色。多巴胺改性的丝素导管置于REDV多肽(100 ml水溶液中加0.1 g REDV多肽)溶液中反应7 h，获得经多巴胺改性并具有REDV多肽的丝素导管。实验导管分组：丝素蛋白支架(SF)、具沟槽结构的丝素蛋白支架(TOPOLOGY)、多肽改性丝素蛋白支架(REDV)、多肽改性且具沟槽结构的丝素蛋白(TOPOLOGY/REDV)。

 2.3. 丝素导管的表征 2.3.1. 扫描电镜观察(SEM)

将材料剪成3 × 4 mm大小的块状薄膜，烘干处理，用导电胶粘至载物台上进行喷金处理，再置于扫描电子显微镜下观察材料内表面的微观形态结构。

 2.3.2. 力学性能实验

采用拉伸试验对不同分子量的丝素导管进行力学性能测定。将浸没在PBS溶液中的丝素导管取出用标尺精确测定丝素导管厚度，每组至少设置3个平行样，室温下将制备的所有样品使用电子万能试验(TFW-58)进行样品的纵向拉伸试验，试验的拉伸速度为10 mm/min。应力–应变曲线和扬氏模量主要用于反映丝素导管的力学特性，测定结束后，将获得最大荷重，最大荷重位移，断裂伸长率，拉伸强度，屈服强度和屈服强度伸长率，然后根据测量数据绘制应力–应变曲线。基于应力–应变曲线的初始部分的斜率计算得到杨氏模量。

 2.4. 细胞实验 2.4.1. 材料预处理及细胞培养

将丝素移植物浸泡于75%酒精中过夜，水洗5次去除酒精，紫外照射2 h，使材料保持内表面朝上，置于24孔板孔底。RSC96细胞(大鼠雪旺细胞)在含10%胎牛血清(FBS)、1%链霉素及青霉素(PS)的DMEM培养液中，置于37℃二氧化碳细胞培养箱培养两天。

 2.4.2. 细胞毒性试验

使用0.25%胰酶消化RSC96细胞，获得细胞悬液，将其以3 × 105/孔种于24孔板内的材料表面，放于37℃二氧化碳培养箱中培养2天，将CCK8试剂(Beyotime Biotechnology)与细胞培养液按照1:10的比例加至孔板各孔，37℃培养箱孵育4 h，使用酶标仪测定其在450 nm处的OD值(吸光度)。其中细胞活力百分比计算公式为：

细胞活力百分比(%) = [A(实验) − A(空白)]/[A(对照) − A(空白)] × 100%

A(实验)：生长于支架材料表面的细胞于CCK-8试剂孵育后所测得的OD值

A(对照)：无支架材料，细胞在完全培养基培中与CCK-8试剂孵育后所测得的OD值

A(空白)：无细胞的完全培养基与CCK-8试剂孵育后所测得的OD值

 2.4.3. 细胞趋向性实验

将0.25%胰酶消化所获得的RSC96细胞悬液以2 × 105/孔种植于24孔板内的材料上，放置于37℃培养箱中，培养1~5天，取1 d，5 d细胞进行鬼笔环肽骨架染色。使用鬼笔环肽试剂染色1 h标记细胞骨架，PBS清洗三次，荧光封片液封片。显微镜下观察细胞在材料上的趋向性及状态。

 2.5. 动物实验

将30只SD大鼠随机分成六组，缺损组(Untreated)、自体组(Control)、丝素蛋白支架组(SF)、具沟槽丝素蛋白支架组(TOPOLOGY)、REDV多肽修饰丝素蛋白支架组(REDV)、REDV修饰且具沟槽结构丝素蛋白支架组(TOPOLOGY/REDV)，进行大鼠坐骨神经缺损桥接模型的制备：大鼠腹腔注射复合麻醉剂(硫酸镁42.4 g，水合氯醛85 g，戊巴比妥钠17.72 g，无水乙醇285 ml，丙二醇676 ml，三蒸水1039 ml 溶解制得)，麻醉后对左侧下肢进行剃毛处理，碘伏消毒，暴露坐骨神经，减去约1 cm的坐骨神经，缺损组不做处理，直接将肌肉与外皮缝合；自体组将缺损两端神经缝合，随即缝合肌肉与外皮；导管组则是将缺损两端神经分别与导管两端缝合，再缝合肌肉与外皮。造模结束12 w后进行心脏灌注，取经手术处理过的一侧大鼠坐骨神经，置于4%多聚甲醛溶液中固定24 h。取出大鼠的腓肠肌及胫前肌，分别称重，根据公式计算以获得靶肌湿重比：靶肌湿重率 = m手术侧靶肌/m正常侧靶肌。

 2.6. 免疫荧光染色

为了观察12 w术后大鼠坐骨神经的恢复状况，将经4%多聚甲醛固定的坐骨神经依次置于5%、10%、30%的蔗糖溶液中浸泡24 h梯度脱水，进行冰冻切片和免疫荧光染色。选择NF (Neurofilament-L (DA2) Mouse mAb)和S100 (S100 Beta Rabbit Polyclonal Antibody)作为一抗试剂分别标记轴突和髓鞘，于4℃环境中染色过夜，PBS清洗三次；二抗分别选用Goat Anti-Mouse IgG H&L (Alexa Fluor 488) preadsorbed、Goat Anti-Rabbit IgG H&L (Cy3) preadsorbed试剂，37℃染色2 h，PBS清洗三次；Hoechst试剂核染15 min，在蔡司显微镜下观察坐骨神经损伤部位染色状况。

 3. 结果与讨论 3.1. 丝素导管的内部形貌

使用扫描电镜观察丝素导管内表面微观形貌。经氯化钠制孔处理，各导管组内表面均可见孔径。其中丝素蛋白支架组(SF)与多肽改性丝素蛋白支架组(REDV)内表面无沟槽结构，呈现平面结构(图1A，图1B)。经表面沟槽处理后，具有沟槽结构的丝素蛋白支架组(TOPOLOGY)和REDV修饰且具有沟槽结构的丝素蛋白支架组(TOPOLOGY/REDV)内表面均清晰可见间距一致具有取向的沟槽结构(图1C，图1D)。

图1. 丝素移植物内表面SEM图，A. 丝素导管组；B. REDV多肽修饰丝素导管组；C. 具沟槽结构丝素导管组；D. REDV多肽修饰且具沟槽结构的丝素导管组

 3.2. 力学表征测试

通过万能拉伸机对丝素导管的拉力测试结果，绘制了应力–应变曲线。丝素蛋白支架组杨氏模量(7.803 ± 0.922 MPa) < REDV多肽修饰丝素蛋白支架组(8.296 ± 4.477 MPa) < 具沟槽结构丝素蛋白支架组(8.858 ± 1.423 MPa) < REDV多肽修饰且具沟槽结构的丝素蛋白支架(9.049 ± 1.434 MPa) (图2B)。SF支架组、REDV支架组、TOPOLOGY支架组、TOPOLOGY/REDV支架组抗压强度分别为1.657 ±0.539 N、1.614 ± 0.766 N、1.441 ± 0.377 N、1.426 ± 0.562 N，缝合强度分别为0.453 ± 0.086 N、0.379 ± 0.099 N、0.225 ± 0.063 N、0.211 ± 0.027 N，与移植物的杨氏模量相比，它们的缝合强度和抗压强度呈现相反趋势(图2C，图2D)。

 3.3. 细胞实验

RSC96细胞种植于材料表面培养1天后使用鬼笔环肽试剂染色，根据染色结果，可观察到细胞在材料表面培养一天后，可清晰观察到细胞胞丝，表明细胞生长状态良好；细胞培养5天后大量增殖，且可明显观察到细胞在具有沟槽结构的TOPOLOGY组及TOPOLOGY/REDV组表面沿着沟槽结构生长(图3c、图3d、图3g、图3h)。

图2. 丝素导管力学表征

图3. 鬼笔环肽细胞骨架染色，标尺为100微米

 3.4. 细胞毒性

使用cck8测定细胞在各材料表面生长的增殖状况，施旺细胞在SF、REDV、TOPOLOGY、TOPOLOGY/REDV各丝素蛋白导管上生长两天时的细胞活力百分比分别为1.096 ± 0.007、1.214 ± 0.134、1.218 ± 0.099、1.303 ± 0.155，均达到了百分百(见图4)，各组间细胞活力百分比无显著性差异。

图4. 2 d细胞活力百分比

 3.5. 动物实验

采用SD大鼠坐骨损伤模型，如图5所示，使用神经移植物进行手术修复10 mm大鼠坐骨损伤。术后12 w，对腓肠肌、胫前肌湿重比进行测量，自体组(control)、丝素蛋白支架组(SF)、REDV修饰的丝素蛋白支架组(REDV)、具沟槽结构的丝素蛋支架组(TOPOLOGY)、REDV修饰且具沟槽结构的丝素蛋白支架组(TOPOLOGY/REDV)胫前肌湿重比分别为0.558 ± 0.074、0.199 ± 0.036、0.242 ± 0.038、0.179 ± 0.0041、0.407 ± 0.034，腓肠肌湿重率分别为0.510 ± 0.058、0.167 ± 0.025、0.212 ± 0.047、0.149 ± 0.038、0.312 ± 0.080 (见图6)，SF组、REDV组、TOPOLOGY组与自体胫前肌湿重比差异具有统计学意义(P < 0.01)，TOPOLOGY/REDV组胫前肌湿重比与自体组相比差异无统计学意义(P > 0.05)。各实验组腓肠肌湿重比与自体组相比，差异具有统计学意义(P < 0.05)。

图5. 丝素导管移植缝合

图6. 术后12W胫前肌、腓肠肌湿重比(*P < 0.05)

 3.6. 免疫荧光染色

术后12W坐骨神经取材进行冰冻切片，使用轴突、髓鞘标记物NF、S100进行免疫荧光染色，其中NF阳性再生轴突被标记为绿色荧光，S100阳性的髓鞘被标记为红色荧光，根据染色结果可以看出损伤神经愈合过程中都具有一定程度的增生现象(图7e-t)。以自体组神经移植物促进损伤神经再生的效果(图e-h)作为检验标准，REDV组(图i-l)及TOPOLOGY组(图m-n)受损神经并未完全愈合，TOPOLOGY/REDV支架组无缺口存在(图q-t)且再生轴突与再生髓鞘的生长状态与自体组相似。

图7. 术后12W大鼠坐骨神经纵切，NF、S100、Hoechst33342免疫荧光标记染色，标尺为 = 200微米

 3.7. 讨论

周围神经可因各组物理因素而受损，例如：自然灾害、运动损伤、交通事故及药物注射受损等，是临床上常见的损伤 [ 17 ] [ 18 ]，神经缺损间隙较短时，临床上一般采取直接缝合修复，当缺损较长时需要使用移植物桥接于缺损神经两端以促进缺损神经愈合 [ 19 ]。自体神经移植是目前公认的神经移植“金标准”，但自体神经来源匮乏，且会对供体组织区域造成二次伤害而应用受限 [ 20 ]。制备合适的神经移植替代物成为目前研究的热点。

丝素蛋白是自然界最丰富且来源最为广泛的生物材料之一，作为天然大分子蛋白它具有生物相容性好，力学性能优良等优点。此外，丝素蛋白还具有可加工性，它可以用作各种形式的生物材料 [ 21 ]。

多巴胺在碱性环境中氧化自聚合，几乎能在任何类型的材料表面形成具有黏附性的聚多巴胺层。聚多巴胺能促进细胞的粘附，具有良好的生物相容性，因此聚多巴胺在神经修复中被广泛应用于各类生物材料支架的表面进行改性 [ 22 ] [ 23 ]。使用活性物质改性修饰丝素蛋白较为困难，因此通过在丝素蛋白移植物表面生成聚多巴胺再共价连接REDV多肽，从而达到活性物质改性修饰丝素蛋白的目的。在组织工程中，合适的微/纳米取向可以指导组织再生过程中细胞的生长和迁移 [ 24 ]。

本研究通过具有均一沟槽的PDMS膜制备丝素导管，在弱碱性环境中使用多巴胺在丝素蛋白支架表面自聚合形成聚多巴胺。Arg-Glu-Asp-Val(REDV)多肽与丝素蛋白支架表面的聚多巴胺结合，从而获得了一种基于精氨酸–谷氨酸–天冬氨酸–缬氨酸修饰的丝素蛋白移植物。

本实验结果表明TOPOLOGY/REDV丝素导管内表面具有均一的取向性沟槽结构，该结构对于细胞的取向生长起到了引导作用，从而促进损伤神经两断端的愈合。较普通丝素导管而言TOPOLOGY/REDV丝素导管的细胞毒性有所降低，力学性能有所改善。同时REDV多肽改性丝素导管内表面的均一沟槽结构对于神经细胞的定向生长具有诱导作用。导管表面的聚多巴胺颗粒及REDV肽有效促进了细胞在材料表面的粘附与扩散。本研究制备的精谷天缬肽改性丝素导管为丝素蛋白在神经损伤再生领域提供了新的应用。

 文章引用

李文慧,姚 珂,张鲁中,杨宇民. 精氨酸–谷氨酸–天冬氨酸–缬氨酸改性丝素导管的制备及性能研究 Study on Preparation and Performance of Arg-Glu-Asp-Val Modified Silk Fibroin Tube[J]. 生物过程, 2021, 11(04): 123-133. https://doi.org/10.12677/BP.2021.114014

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