【作者】 王健；

【导师】 王琳；

【作者基本信息】 华中科技大学 ， 临床检验诊断， 2017， 博士

【摘要】 第一部分:制备神经保护性丝胶水凝胶作为细胞载体应用于中风修复并阐述了其机制目的:合成一种具有神经保护性的生物水凝胶应用于中风修复并对其进行评估方法:首先通过LiBr抽提法从蚕茧中获取丝胶液;然后选用Genipin作为交联剂,通过共价交联反应,合成丝胶水凝胶。我们对该丝胶水凝胶的二级结构,机械性能,吸水膨胀率,微观结构,降解性进行评估;通过原代神经元细胞的形态和细胞毒性实验检测了该水凝胶的细胞相容性;通过OGD模型评估丝胶水凝胶的神经保护作用;通过原代神经元轴突生长实验评价丝胶水凝胶的神经营养性;通过半定量,WB和信号通路抑制剂实验来揭示潜在的分子机制;通过体内移植实验评估该水凝胶递送细胞的能力。结果:合成的丝胶水凝胶拥有低的机械性能,稳定的吸水膨胀率,多孔的微观结构以及很好的降解性。细胞毒性实验显示其具备很好的细胞相容性,其对缺氧条件下的神经元具有的保护作用也在OGD模型中被证明。在0.05mg/mL的浓度下,丝胶液能很好的促进原代神经元轴突的生长。蛋白印记实验证明了神经保护性是通过Bcl-2/Bax信号通路发挥作用的,通路抑制剂实验证明神经营养作用是通过Lkb-1/Nuak-1通路发挥作用的。水凝胶搭载细胞体内移植实验证明了该水凝胶可以作为一个合格的细胞载体应用在组织工程修复中。结论:合成的京尼平交联的丝胶水凝胶可以作为一个理想的生物材料应用于中风的组织工程治疗策略中。第二部分:具有可注射性和导电性的丝胶支架用于中枢神经组织的修复目的:制作一种具有可注射性和导电性的碳纳米管丝胶支架并对其性质进行评估方法:首先通过温和LiBr抽提法从蚕茧中获取丝胶液;然后运用超声的方式在未成胶的丝胶液中充分混匀碳纳米微管,之后使用共价交联的方式制备丝胶水凝胶并冻干获得支架。我们对该丝胶支架的物理和化学性质进行深入的研究,包括它的微观结构,机械性能,吸水膨胀行为;通过共聚焦分析,CCK-8实验以及流式细胞染色检测了该丝胶支架的细胞相容性;通过体外和体内注射实验探索该丝胶支架的可注射性质;通过电阻测量实验来评估它的导电性能;通过Tuj-1免疫荧光,实时定量PCR,以及蛋白印记实验来评估它的促BMSCs分化能力。结果:我们成功地制备了这种碳纳米微管的丝胶支架,共聚焦分析发现碳纳米微管可以均匀地分散在支架内。电子扫描显微镜的结果显示该丝胶支架含有大量的,互相连通的微孔结构。压缩模量实验发现,该丝胶支架的机械模量是可以通过调整碳纳米微管的掺入量来进行调控。吸水膨胀率实验表明,在PBS溶液中浸泡48小时后,该丝胶支架的吸水膨胀率就能稳定下来。在细胞毒性实验中,我们发现掺入0.1-0.5mg/mL碳纳米微管的丝胶支架具备非常优越的细胞相容性。在体内和体外的注射实验中,我们证明了这种丝胶支架的可注射性。受益于碳纳米微管的掺入,该丝胶支架的电导性有显著的提高,而且它跟碳纳米微管的含量有明显的相关性。Tuj-1免疫荧光,实时定量PCR,以及蛋白印记实验证明了该丝胶支架可以促进骨髓间充质干细胞向神经源性细胞分化。结论:这种具有可注射性,导电性以及促分化的碳纳米微管丝胶支架有很大的潜力应用于中枢神经组织修复领域。更多还原

【Abstract】 Part 1.A neuro-protective sericin hydrogel explored as a cell carrier for the repair of strokeObjective:Fabricate a neuro-protective sericin hydrogel for the repair of stroke and evaluate its performance.Methods:We obtained the sericin solution from the cocoons by the LiBr extraction method.The genipin was selected as a crosslinker,and the genipin crosslinked sericin hydrogel was fabricated through a covalent cross-linking reaction.A serial of properties were evaluated,including the secondary structure,the mechanical strength,the swelling ratio,the microstructure and the degradation behaviors.The cytoskeleton staining and the Live&Dead staining were performed to study the cyto-compatibility of the sericin hydrogel.The neuro-protective property was assessed by the OGD model and the neurotrophic effect was assessed by the primary cortical neuronal axon outgrowth experiment.The underlying molecular mechanism was explored by the western blotting and the signal pathway inhibition assay.The in vivo transplantation was also performed to evaluate its performance as a cell carrier.Results:The sericin solution formed a stable and light blue hydrogel.It exhibits the lower mechanical strength,the stable swelling ratio,the porous microstructure and the good degradation behavior.The F-actin staining and Live&Dead staining have confirmed its excellent cyto-compatibility.The OGD assay has found the neuro-protective property of the sericin solution and its degraded products.Under 0.05mg/mL sericin solution,the outgrowth of neuronal axon was enhanced.The western blotting assay confirmed that the Bcl-2/Bax signaling pathway play a key role in the neuro-protective behavior.The Nuak-1 inhibitor assay showed the enhanced outgrowth of neuronal axon benefits from the Lkb-1/Nuak-1 pathway.The in vivo transplantation experiment indicated that this sericin hydrogel have the powerful potential in the neural tissue engineering.Conclusion:Our fabricated genipin crosslinked sericin hydrogel could be utilized as a promising biomaterial in the neural tissue engineering for the stroke treatment.Part 2.A carbon nano-tube hybrid sericin scaffold with injectability and conductivity characteristics applied in neural repairing field.Objective:Design a conductive sericin hydrogel with injectability and utilize it in neural tissue repair fieldMethods:The gentle LiBr extraction method was used to obtain the pure sericin solution.The carbon nano-tubes were mixed in the genipin crosslinked sericin hydrogels by the ultrasonic method.The physical and chemical characteristics of the carbon nano-tube hybrid sericin scaffold were tested,including the porous micro-structure,the mechanical modulus and the swelling behaviors.The confocal analysis,the CCK-8 assay and the flow cytometry were performed to evaluate its cyto-compatibility.The injectability property of the hybrid sericin scaffold was evaluated by the in vitro and in vivo injection assay.The electrical property was study by the impedance measurement experiment.The immune-fluorescence staining,the Real-time quantitative PCR and the western blotting experiments were performed to study the pro-differentiation characteristics of this hybrid sericin scaffold.Results:The CNTs hybrid sericin scaffold was successfully synthesized and the CNTs were homogeneously distributed in the scaffold.The SEM images confirmed that the scaffold possessed the interconnected porous microstructures.We also found that the mechanical modulus of the scaffold was controllable through adjusting the content of the CNTs during the synthesis process.The swelling assay showed us that the scaffold had the stable swelling ratio after it was immersed in PBS for 48 hours.In the cyto-toxicity assay,the scaffolds with 0.1-0.5mg/mL CNTs exhibited the excellent cyto-compatibility.In the in vitro and in vivo injection experiments,we found and confirmed that the scaffold possessed the injectable property.Benefiting from the incorporated CNTs,the impedance of the scaffold was significantly decreased.In the Real-time quantitative PCR,the Tuj-1 immune-fluorescence staining and the western blotting experiments,we found that the scaffold with CNTs can promote the BMSCs differentiated into neural cells.Conclusion:This CNTs hybrid sericin scaffold with injectability and pro-differentiation properties has the potential in the neural tissue repair field.更多还原