脊髓损伤修复的复合透明质酸水凝胶支架的构建及其评价

发布时间：2018-03-04 14:19

本文选题：腺病毒　切入点：β-神经生长因子　出处：《山东大学学报(医学版)》2017年09期 　论文类型：期刊论文

【摘要】：目的评价接枝Nogo-66受体(NgR)抗体和多聚左旋赖氨酸(PLL)的透明质酸(HA)水凝胶支架的作用,及转染β神经生长因子(β-NGF)基因的内皮祖细胞(EPCs)能否在支架内稳定表达外源基因。方法采用冰冻干燥法制备多孔HA水凝胶材料,将NgR抗体和PLL接枝到HA支架上。体外培养EPCs,并通过携带β-NGF腺病毒转染EPCs,将EPCs与支架共培养,通过扫描电镜观察支架结构,将细胞按处理方式及培养方式分为β-NGF+组、Scaffold-β-NGF+组、β-NGF-组、Scaffold-β-NGF-组、对照组和Scaffold-对照组。采用细胞活力(CCK-8)法及HE法检测EPCs在支架上的生长情况,采用乳酸脱氢酶(LDH)-细胞毒性法检测支架的细胞毒性,采用ELISA法、Rt-PCR法和Western blotting法检测β-NGF的表达。结果制备的支架具备三维多孔结构,孔径(200±15)μm,大小较均一,EPCs在支架上的生长明显优于培养板中的生长状况(F=468 518.044,P0.001),且随时间推移,细胞数目差异有统计学意义(F=2 678 658.138,P0.001),支架的细胞毒性相比培养板无明显异常(F=0.680,P=0.429)。EPCs在支架上可以稳定表达β-NGF,且Scaffold-β-NGF+组mRNA及蛋白质的表达量均优于β-NGF+组(mRNA:F=651.554,P0.001;蛋白:F=14 671.733,P0.001)。结论 EPCs与NgR抗体-PLL复合A水凝胶支架具有良好的生物相容性。该HA水凝胶支架有望作为修复脊髓损伤的组织工程载体。
[Abstract]:Objective to evaluate the effect of graft Nogo-66 receptor (Nogo-66 receptor) antibody and poly (L-lysine) (PLL) hydrogel scaffold on hyaluronic acid (HA). Whether the endothelial progenitor cells transfected with 尾 -NGF gene could stably express foreign genes in the scaffold. Methods porous HA hydrogel materials were prepared by freeze drying method. NgR antibody and PLL were grafted onto HA scaffold. EPCs were transfected with 尾 -NGF adenovirus and co-cultured with EPCs. The stents were observed by scanning electron microscope. The cells were divided into 尾 -NGF group (Scaffold- 尾 -NGF group), 尾 -NGF- group (Scaffold- 尾 -NGF- group) and 尾 -NGF group (Scaffold- 尾 -NGF- group). In control group and Scaffold- control group, cell viability CCK-8 and HE methods were used to detect the growth of EPCs on the scaffold, and the cytotoxicity of the stents was detected by lactate dehydrogenase (LDH) -cytotoxicity assay. The expression of 尾 -NGF was detected by ELISA Rt-PCR and Western blotting. Results the scaffolds had a three-dimensional porous structure with a pore diameter of 200 卤15 渭 m. The growth of EPCs on the scaffold was obviously better than that in the culture plate. There was significant difference in the number of cells. Compared with the culture plate, the cytotoxicity of the scaffold was not abnormal. MRNA and protein expression in Scaffold- 尾 -NGF group were significantly higher than those in 尾 -NGF group. Conclusion the expression of 尾 -NGF in Scaffold- 尾 -NGF group is higher than that in 尾 -NGF group. Conclusion the expression of 尾 -NGF in Scaffold- 尾 -NGF group is higher than that in 尾 -NGF group. Conclusion the expression of mRNA and protein in Scaffold- 尾 -NGF group is higher than that in 尾 -NGF group. Conclusion the expression of 尾 -NGF in Scaffold- 尾 -NGF group is higher than that in 尾 -NGF group. The HA hydrogel scaffold has good biocompatibility with NgR antibody PLL composite A hydrogel scaffold which is expected to be used as tissue engineering carrier for spinal cord injury repair.
【作者单位】：山东大学第二医院脊柱外科;临邑县人民医院骨科;
【基金】：山东省科技发展计划(2014GSF118045)
【分类号】：R651.2

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