负载虎杖苷的胶原/硫酸肝素支架修复大鼠脊髓损伤  

Polydatin loaded collagen/heparin sulfate scaffold in the treatment of spinal cord injury in rats

作　　者：刘康 王勇平[2] 左可斌 杨海涛 贾斌 Liu Kang;Wang Yongping;Zuo Kebin;Yang Haitao;Jia Bin(First Department of Orthopedics,Qingyang People’s Hospital,Qingyang 745000,Gansu Province,China;Department of Orthopedics,First Hospital of Lanzhou University,Lanzhou 730000,Gansu Province,China)

机构地区：[1]甘肃省庆阳市人民医院骨一科,甘肃省庆阳市745000 [2]兰州大学第一医院骨科,甘肃省兰州市730000

出　　处：《中国组织工程研究》2022年第28期4500-4506,共7页Chinese Journal of Tissue Engineering Research

基　　金：甘肃省自然科学基金(20JR5RA369),项目负责人:王勇平。

摘　　要：背景:目前关于虎杖苷神经保护作用的研究逐渐成为神经科领域关注的热点,研究方向多集中在缺血性脑血管病方面,在脊髓损伤修复中应用的研究较少。目的:以胶原/硫酸肝素支架为载体,将虎杖苷局部应用于脊髓损伤部位,观察修复效果。方法:①制备胶原/硫酸肝素支架与负载0.5,1,1.5 mmol/L虎杖苷的胶原/硫酸肝素支架,将第3代大鼠神经干细胞分别接种于4种支架上,采用CCK-8法检测细胞增殖情况;在诱导神经干细胞的神经向分化过程中,采用免疫荧光染色与RT-PCR检测胶质纤维酸性蛋白、Tuj-1和Oligo的表达。②建立成年雄性SD大鼠半横切脊髓损伤模型,分3组,模型对照组(n=10)不植入任何材料,对照组(n=10)植入胶原/硫酸肝素支架,实验组(n=10)植入1 mmol/L虎杖苷/胶原/硫酸肝素支架;同时设立假手术组(n=10)。术后8周内,利用BBB评分测试大鼠右后肢运动功能;术后第8周取材,进行脊髓组织学观察、免疫组化分析与Western Blot检测。结果与结论:①培养第3,7天时,负载1 mmol/L虎杖苷支架上的细胞增殖吸光度值高于其他3组支架(P<0.05)。②诱导7 d后的免疫荧光染色显示,负载1,1.5 mmol/L虎杖苷支架组的胶质纤维酸性蛋白表达少于其他两组,Oligo与Tuj-1表达多于其他两组。③RT-PCR检测结果显示,负载1,1.5 mmol/L虎杖苷支架组的胶质纤维酸性蛋白mRNA表达水平低于其他两组(P<0.05),4组间的Tuj-1 mRNA表达水平无明显差异,负载1 mmol/L虎杖苷支架组的Oligo mRNA表达水平高于其他3组(P<0.05)。④脊髓损伤修复实验显示,实验组术后2-8周的BBB评分始终高于模型对照组与对照组(P<0.05);苏木精-伊红染色显示,对照组与观察组脊髓缺损部位被支架填充,组织间的间隙小于模型对照组,其中观察组的组织连续性与组织间隙改善好于对照组;免疫组化与Western Blot检测显示,实验组的神经丝蛋白200的表达高于对照组、模型对�BACKGROUND:At present,the research on the neuroprotective effect of polydatin has gradually become a hot spot in the field of neurology.The research direction is mostly focused on ischemic cerebrovascular disease,and there are few studies on the application of spinal cord injury.OBJECTIVE:With collagen/heparin sulfate scaffold as carrier,polydatin was applied to the injured spinal cord to observe the repair effect.METHODS:(1)Collagen/heparin sulfate scaffolds and collagen/heparin sulfate scaffolds loaded with 0.5,1,1.5 mmol/L polydatin were prepared.The third generation of rat neural stem cells was seeded on four kinds of scaffolds,and the proliferation of cells was detected by CCK-8 assay.During inducing neural differentiation of neural stem cells,the expression levels of glial fibrillary acidic protein,Tuj-1 and Oligo were detected by immunofluorescence staining and RTPCR.(2)The spinal cord injury model of adult male SD rats was established and divided into three groups.The model control group(n=10)was not implanted with any materials.The control group(n=10)was implanted with collagen/heparin sulfate scaffold,and the experimental group(n=10)was implanted with 1 mmol/L polydatin/collagen/heparin sulfate scaffold.Meanwhile,sham operation group(n=10)was set up.BBB score was used to test the motor function of the right hind limb within 8 weeks after operation.At 8 weeks after operation,the spinal cord tissues were taken for histological observation,immunohistochemical analysis,and western blot assay.RESULTS AND CONCLUSION:(1)At 3 and 7 days of culture,the absorbance value of cell proliferation on 1 mmol/L polydatin/collagen/heparin sulfate scaffold was higher than that on the other three scaffolds(P<0.05).(2)Immunofluorescence staining 7 days after induction showed that the expression of glial fibrillary acidic protein in 1 and 1.5 mmol/L polydatin/collagen/heparin sulfate scaffold groups were less than those in the other two collagen/heparin sulfate scaffold groups,but the expression levels of Oligo and Tuj-1 were

关 键 词：虎杖苷 胶原 硫酸肝素 支架 组织工程支架 生物材料 脊髓修复 脊髓损伤 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]