用于脊髓损伤修复的NgR抗体-PLL复合HA水凝胶支架的构建及其评价

发布时间：2018-04-21 10:05

本文选题：腺病毒 + β-神经生长因子　；参考：《山东大学》2017年硕士论文

【摘要】：目的神经元细胞是人体内再生能力最差的细胞之一,其构成的神经系统亦是人体内至关重要的系统之一,因此神经元细胞发生损伤往往会导致严重的不可逆的后果[1,2]。如何实现神经元细胞损伤后神经细胞的修复、再生直至神经系统功能恢复一直是相关学科的研究重点及难点[3-6]。本实验将组织工程学、转基因技术及干细胞治疗技术有机结合,为修复脊髓损伤(SCI)提供了新的方向[7,8]。成功修复脊髓损伤的关键在于:①种子细胞的选择和培养;②具有理想生物相容性的三维立体框架材料的构建[9-19];③有助于神经恢复的微环境的创建。本课题选择神经元细胞细胞基质主要构成之一透明质酸(HA)作为三维立体框架主要原材料,制造该HA水凝胶复合支架,将可以稳定分泌β-神经生长因子(β-NGF)的内皮祖细胞(EPCs)与接枝Nogo-66受体抗体(NgR抗体)和多聚左旋赖氨酸(PLL)的HA支架共培养,评价转染了β-NGF基因的EPCs与嫁接NgR抗体以及PLL的HA支架的生物相容性,研究该HA支架作为修复SCI的组织工程学框架载体的可能性,为通过有机结合组织工程学及干细胞治疗技术治疗脊髓损伤等疾病提供研究基础。方法以冰冻干燥方法制备多孔HA水凝胶框架材料,将NgR抗体和PLL通过化学接枝法接枝到HA水凝胶支架上。提取EPCs,体外培养,取第三代EPCs用于试验研究,并通过携带β-NGF基因的腺病毒将其转染,然后将EPCs与支架共培养。通过扫描电镜观察HA水凝胶的内部结构,通过细胞活力检测(CCK-8)及HE染色法观察EPCs在支架上的生长情况,通过检测支架/细胞复合物乳酸脱氢酶释放量评价HA水凝胶支架材料细胞毒性,通过酶联免疫吸附实验(ELISA)、实时定量荧光PCR(Real Time-PCR)、蛋白免疫印迹法(Western blot)评估β-NGF基因在体外的表达、释放情况。结果成功培养出EPCs,其生长良好、增值活跃,腺病毒携带β-NGF转染EPCs后其可以稳定表达β-NGF。制造的HA水凝胶支架具备理想的三维孔状结构,EPCs在支架上生长良好,且明显优于EPCs在培养板内生长,HA水凝胶支架的细胞毒性较培养板相比无明显差异,且β-NGF在支架上表达与对照组相比存在明显的优势。结论转染了β-NGF的EPCs生长旺盛并且能够稳定表达β-NGF,可以作为神经组织工程学的种子细胞。EPCs可以在接枝NgR抗体和PLL的透明质酸水凝胶支架上稳定增值、表达,说明EPCs与接枝NgR抗体和PLL的透明质酸水凝胶支架有良好的生物相容性。该HA支架可以作为修复SCI的组织工程学载体。
[Abstract]:Objective Neuron cell is one of the worst regenerative cells in human body, and its nervous system is also one of the most important systems in human body. Therefore, neuronal cell damage often leads to serious irreversible consequences [1 / 2]. How to repair and regenerate the nerve cells after neuronal cell injury, until the function of the nervous system is restored, has always been the focus and difficulty of related disciplines [3-6]. In this study, tissue engineering, transgenic technology and stem cell therapy were combined to provide a new direction for the repair of spinal cord injury (SCI). The key to the successful repair of spinal cord injury lies in the selection of seed cells of 1 / 1 and the construction of 3 dimensional frame material with ideal biocompatibility [9-19] 3 which is helpful to the establishment of microenvironment for nerve recovery. In this study, hyaluronic acid (HA), one of the main components of neuronal cell matrix, was used as the main raw material of three-dimensional framework to fabricate the HA hydrogel composite scaffold. Endothelial progenitor cells (EPCs), which secrete 尾 -NGF stably, were co-cultured with grafted Nogo-66 receptor antibody (NgR) and poly (L-lysine) (PLL) HA scaffold. To evaluate the biocompatibility of EPCs transfected with 尾 -NGF gene and grafted NgR antibody and HA scaffold of PLL, and to study the possibility of using the scaffold as a tissue engineering framework carrier for SCI repair. To provide a basis for the treatment of spinal cord injury by organically combining tissue engineering and stem cell therapy. Methods porous HA hydrogel frame material was prepared by freeze drying method. NgR antibody and PLL were grafted onto HA hydrogel scaffold by chemical grafting method. EPCs were extracted and cultured in vitro. The third generation of EPCs was used for the experimental study. The EPCs was transfected by adenovirus carrying 尾 -NGF gene, and then co-cultured with the scaffold. The internal structure of HA hydrogel was observed by scanning electron microscope (SEM), the growth of EPCs on the scaffold was observed by cell viability test (CCK-8) and HE staining. The cytotoxicity of HA hydrogel scaffold was evaluated by detecting the release of lactate dehydrogenase from scaffold / cell complex. The expression of 尾 -NGF gene in vitro was evaluated by enzyme-linked immunosorbent assay (Elisa), real-time quantitative fluorescence PCR(Real Time-PCRN and Western blotting. Release. Results EPCs were cultured successfully, and their growth was good and the proliferation was active. Adenovirus carrying 尾 -NGF could stably express 尾 -NGF after transfection into EPCs. The HA hydrogel scaffolds had ideal three-dimensional pore structure. EPCs grew well on the scaffold, and the cytotoxicity of HA hydrogel scaffolds was significantly better than that of EPCs in culture plate. The expression of 尾-NGF on the scaffold was significantly superior to that of the control group. Conclusion EPCs transfected with 尾 -NGF has a strong growth and stable expression of 尾 -NGF, which can be used as seed cells of neural tissue engineering. EPCs can be expressed on the scaffold of NgR antibody and PLL hydrogel with hyaluronic acid. The results showed that EPCs had good biocompatibility with NgR antibody and PLL hydrogel scaffold. The HA scaffold can be used as a tissue engineering carrier for SCI repair.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

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