超分子水凝胶用于构建髓核细胞支架的可行性研究

发布时间：2018-04-01 11:07

  本文选题：超分子水凝胶　切入点：组织工程　出处：《南京大学》2017年硕士论文

【摘要】：背景:椎间盘退变性疾病是临床的常见疾病,目前的临床治疗方法无法达到在结构和功能层面上修复病变椎间盘。髓核组织工程运用具备良好功能的髓核种子细胞来替代已经发生退变的髓核细胞,由此恢复正常的髓核生理构造和功能,逆转椎间盘退变过程,成为了治疗椎间盘退变的一个研究方向。在髓核组织工程研究中,细胞支架对种子细胞的活性和功能表达至关重要。目前大多选用天然材料水凝胶或者是人工合成的高分子聚合物水凝胶,这两类材料均有其自身的缺点。随着材料化学的发展,一类以小分子寡肽为基本单位的超分子水凝胶因为具备优秀的生物相容性,良好的力学性能和近似细胞外基质的结构有望成为用于构建髓核组织工程细胞支架的新的选择。目的:探究以寡肽(Nap-F-F-Y(P))为基本单位复合硫酸软骨素的超分子水凝胶用于构建髓核组织工程细胞支架的可行性。方法:在化学实验室运用固相合成法合成寡肽(Nap-F-F-Y(P)),质谱分析鉴定产物。在碱性磷酸酶作用下发生自组装并与硫酸软骨素复合成胶,探讨不同浓度水凝胶的成胶时间,宏观形态,微观结构以及力学性质等材料学表征。取12周龄大鼠尾椎椎间盘髓核细胞原代培养作为种子细胞,超分子水凝胶复合髓核细胞共培养两周,以不加入水凝胶的普通培养为对照,行细胞活死染色,CKK-8检测观察髓核细胞活性,细胞免疫荧光染色观察髓核细胞特征表型Ⅱ型胶原及聚集蛋白聚糖(Aggrecan)的表达情况。结果:合成的寡肽经质谱分析后证明结构分子量正确,产物纯度较高,可进一步运用于自组装成胶,其成胶浓度范围为0.5%(w/v)至1.5%(w/v),肉眼可见凝胶透明度随浓度增高而降低,寡肽浓度为1%时,成胶速度最快,宏观形态结构最稳定。透射电镜观察水凝胶微观结构可见纳米纤维排列纵横交错,形成纳米级别的孔隙,组成了凝胶内部疏松多孔结构。水凝胶应变及频率动态扫描发现其力学性质良好,具备组织工程水凝胶粘弹性要求。对于1%浓度的寡肽,硫酸软骨素复合后能进一步提升其力学性质。大鼠髓核细胞提取经原代培养后形态均一,类似软骨细胞,表型鉴定显示Ⅱ性胶原及Aggrecan表达完好。经水凝胶培养2周后,活死细胞染色提示髓核细胞活性与普通培养无差异,CKK-8试验提示细胞增殖未受到抑制,细胞免疫荧光发现髓核细胞表型完整。结论:以寡肽(Nap-F-F-Y(P))为基本单位自组装成胶并复合硫酸软骨素的超分子水凝胶在微观结构上近似细胞外基质,具有良好的粘弹性,生物相容度高,与髓核细胞共培养后能维持其细胞活性和表型,且成胶简便,符合生理环境,用于髓核组织工程细胞支架的构建具有较好的可行性。
[Abstract]:Background: disc degeneration is a common clinical disease. The current clinical treatment methods cannot repair the diseased intervertebral disc at the structural and functional levels. The nucleus pulposus tissue engineering uses the nucleus pulposus seed cells with good function to replace the degenerated nucleus pulposus cells. Thus restoring normal physiological structure and function of nucleus pulposus and reversing the process of disc degeneration have become a research direction in the treatment of disc degeneration. Cell scaffolds are crucial to the activity and functional expression of seed cells. At present, natural hydrogels or synthetic polymer hydrogels are mostly used, both of which have their own disadvantages. With the development of material chemistry, A class of supramolecular hydrogels based on small molecular oligopeptides because of their excellent biocompatibility. Good mechanical properties and similar extracellular matrix structure are expected to be a new choice for the construction of scaffolds for tissue engineering cells of nucleus pulposus. Objective: to explore the supramolecular hydrogel with Nap-F-F-YPU as the basic unit and chondroitin sulfate as the basic unit. Methods: Nap-F-F-YPX oligoseptide was synthesized in a chemical laboratory by solid phase synthesis, and the product was identified by mass spectrometry. Self-assembly occurred under the action of alkaline phosphatase and was combined with chondroitin sulfate to form glue. The gelation time, macroscopic morphology, microstructure and mechanical properties of hydrogels with different concentrations were studied. The primary culture of nucleus pulposus cells of rat tail vertebrae at 12 weeks of age was used as seed cells. Supramolecular hydrogel combined with nucleus pulposus cells were cultured for two weeks. The activity of nucleus pulposus cells was detected by CKK-8 staining without hydrogel. Cell immunofluorescence staining was used to observe the expression of type 鈪，

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