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

doi:10.6040/j.issn.1671-7554.0.2017.061

摘要/Abstract

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, P<0.001),且随时间推移,细胞数目差异有统计学意义(F=2 678 658.138, P<0.001),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, P<0.001;蛋白: F=14 671.733, P<0.001)。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。

关键词: 透明质酸水凝胶支架, 腺病毒, β-神经生长因子, 内皮祖细胞, 脊髓损伤

Abstract: Objective To evaluate the role of hyaluronic acid(HA)scaffold modified with Nogo-66 receptor(NgR)antibody and poly-l-lysine(PLL), and to determine whether endothelial progenitor cells(EPCs)transfected with β nerve growth factor(β-NGF)could stably express exogenous gene in stent. Methods The NgR antibody and PLL were grafted to the porous HA hydrogel scaffold which was produced by freeze drying method. In vitro study, the EPCs were isolated from Wistar rats, and then β-NGF was transfected to EPCs by recombinant adenovirus. EPCs were then cultured on the HA hydrogel scaffold, and the internal structure of the HA hydrogel scaffold was observed with scanning electron microscope. EPCs were divided into β-NGF⁺ group, Scaffold-β-NGF⁺ group, β-NGF^- group, Scaffold-β-NGF^- group, control group and Scaffold-control group. The growth of the EPCs cultured on the scaffold was determined with cell vitality detection(CCK-8)and HE. The cytotoxicity of HA hydrogel scaffold was detected with lactate dehydrogenase(LDH). The expression of β-NGF was detected with Rt-PCR, Western blotting and enzyme-linked 山东大学学报 (医学版)55卷9期 -刘东,等.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 \=-immunosorbent(ELISA)assays in vitro. Results The aperture size of the porous three-dimensional structure of HA hydrogel scaffold was(200±15)μm. The EPCs grew better on the scaffold(F=468 518.044, P<0.001). There existed significant difference over time in the number of cells(F=2 678 658.138, P<0.001). There was no difference in cytotoxicity between HA scaffold and culture plate(F=0.680, P=0.429). EPCs could stably express β-NGF on the scaffold, and the mRNA and protein expressions of β-NGF in the Scaffold-β-NGF⁺ group were better than those in the β-NGF⁺ group(mRNA: F=651.554, P<0.001; β-NGF: F=14 671.733, P<0.001). Conclusion EPCs and the newly created HA hydrogel scaffold modified with NgR antibody and PPL have a good biocompatibility. This scaffold may serve as an optimal biomaterial for tissue engineering in spinal cord injury repair.

Key words: EPCs, Spinal cord injury, Adenovirus, β-NGF, Hyaluronic acid hydrogel scaffold

中图分类号:

R681

刘东,朱冬昀,彭长亮,张程,赵杰,高春正. 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[J]. 山东大学学报（医学版）, 2017, 55(9): 53-59.

LIU Dong, ZHU Dongyun, PENG Changliang, ZHANG Cheng, ZHAO Jie, GAO Chunzheng. Construction and appraisal of hyaluronic acid hydrogel scaffold in the treatment of treat spinal cord injury[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2017, 55(9): 53-59.

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