聚乳酸-症基乙酸共聚物/氧化石墨烯纳米纺丝膜搭载脑源性神经营养因子对脊髓损伤的修复作用  

Effect of polylactic-co-glycolic acid/graphene oxide nanofibers combined with brain derived neurotrophic factor on spinal cord injury repair

作　　者：潘肃[1] 齐治平[1] 郑爽[1] 马悦 付川[1] 孔维健 余双奇 杨小玉[1] 张卓[3] Pan Su;Qi Zhiping;Zheng Shuang;Ma Yue;Fu Chuan;Kong Weijian;Yu Shuangqi;Yang Xiaoyu;Zhang Zhuo(Department of Orthopedic Surgery,Second Hospital of Jilin University,Changchun 130022,China;Department of Gynecology,First Hospital of Jilin University,Changchun 130012,China;Department of Orthopedic Surgery,Third Hospital of Jilin University,Changchun 130031,China)

机构地区：[1]吉林大学第二医院骨科,长春130022 [2]吉林大学第一医院妇科,长春130012 [3]吉林大学中日联谊医院,长春130031

出　　处：《中华创伤杂志》2019年第7期597-604,共8页Chinese Journal of Trauma

基　　金：国家自然科学基金(31572217);吉林省教育厅“十三五”科学研究规划(JJKH20180224KJ).

摘　　要：目的探讨搭载有脑源性神经营养因子(BDNF)的聚乳酸-轻基乙酸共聚物(PLGA)/氧化石墨烯(GO)纳米纺丝纤维膜对神经干细胞(NSCs)增殖分化的影响及其对脊髓损伤的修复作用。方法制备PLGA/G0纳米纺丝膜并物理吸附BDNF,采用扫描电镜观察PLGA/GO纺丝膜的微观结构。采用ELISA测定BDNF在PLGA/GO纺丝膜上的搭载效率和释放曲线。将NSCs接种在PLGA/GO和PLGA/GO/BDNF材料表面,釆用MTT法对各组的吸光度值进行测定,利用免疫荧光和PCR对33微管蛋白(Tuj-1)表达进行观察。随后将30只雌性SD大鼠按随机数字表法分为损伤对照组(10只)、PLGA/GO组(10只)和PLGA/GO/BDNF组(10只),利用维纳斯剪切割T,脊髓组织建立脊髓半切损伤动物模型,PLGA/GO组和PLGA/GO/BDNF组分别将对应的纳米纺丝膜原位移植覆盖于损伤处。造模后1,7,14和28d利用BBB评分对大鼠的运动功能进行评价。于伤后1个月时取材,免疫荧光染色观察神经元特异性核蛋白(NeuN)和胶质纤维酸性蛋白(GFAP)的表达情况。结果扫描电镜结果显示,PLGA/GO纺丝膜为不规则的光滑纤维样结构,纤维直径(987.5±176.3)nm,NSCs可以在纺丝膜上正常分化为神经元。ELISA结果显示,PLGA/GO纺丝膜对BDNF的负载率约为47.5%;释放曲线显示,BDNF在第1天首先释放30%左右,随后至第21天释放至60%左右。MTT和PCR结果显示,PLGA/GO/BDNF组的吸光度(A)值和Tuj-1表达显著高于PLGA/G0组(P<0.05)。造模后28d,PLGA/GO/BDNF组BBB评分为(15.3±0.7)分,显著高于损伤对照组的(11.8±0.8)分和PLGA/GO组的(12.7±0.8)分(P<0.05)。免疫荧光结果显示,PLGA/GO/BDNF组NeuN表达为(13.7±2.2)个,显著高于损伤对照组的(4.3±2.9)个(P<0.05);PLGA/GO/BDNF组GFAP表达为(25.6±4.3)%,显著低于损伤对照组的(38.5±6.2)%和PLGA/GO组的(36.7±7.3)%(P<0.05)。结论搭载有BDNF的PLGA/GO纳米纺丝纤维膜可以在体外有效促进NSCs增殖和神经元向分化,并通过体内损伤处原位移植有效修复脊髓�Objective To investigate the effect of polylactic-co-glycolic acid(PLGA)/graphene oxide(GO)nanofibers combined with brain derived neurotrophic factor(BDNF)on neural stem cells(NSCs)proliferation and differentiation as well as on the spinal cord injury repair.Methods PLGA/GO nanofibers were manufactured and absorbed with BDNF,and the microstructure of PLGA/GO nanofibers was observed by scanning electron microscope.The loading efficiency and release curve of BDNF on PLGA/GO nanofibers were measured by ELISA.NSCs were implanted on the surface of PLGA/GO and PLGA/GO/BDNF nanofibers.The absorbance values of each group were measured by MTT method,and the expression of Tuj-1 was observed by immunofluorescence and PCR.A total of 30 female SD rats were divided into control group(n=10),PLGA/GO group(n=10)and PLGA/GO/BDNF group(n=10)according to random number table.T9 spinal cord tissue was cut by Venus scissors to establish spinal cord hemisection injury model of rats.PLGA/GO and PLGA/GO/BDNF nanofibers were implanted onto the surface of injury site.BBB score was used to assess the motion functional recovery of the rats at 1,7,14 and 28 days after operation.Immunofluorescence staining of neuron specific nucleoprotein(NeuN)and glial fibrillary acidic protein(GFAP)were performed to observe the expressions of neurons and astrocytes at the injured site respectively one month after injury.Results The PLGA/GO nanofibers showed an irregular smooth fiber-like structure,and the average fiber diameter was(987.5±176.3)nm.NSCs could differentiate into neurons on the nanofibers.The result of ELISA showed loading rate of BDNF on PLGA/GO nanofibers was about 47.5%.The release curve showed that BDNF was first released about 30%on the first day and then about 60%on the 21st clay.The results of MTT and PCR showed that optical density value and Tuj-1 gene expression in the PLGA/GO/BDNF group were significantly higher than those in the PLGA/GO group(P<0.05).The animal experiment results showed that the BBB score of PLGA/GO/BDNF group was(15.

关 键 词：脊髓损伤 神经干细胞 脑源性神经营养因子 聚乳酸-径基乙酸共聚物/氧化石墨烯 

分 类 号：R651.2[医药卫生—外科学]