巯基聚乙二醇修饰GNPs/miR-29纳米微粒的制备及细胞相容性研究  

Characterization of PEG-SH modified GNPs/miR-29 nanoparticles and cytocompatibility

作　　者：万俊明[1] 朱爽 谢小波[2] 林昭伟[2] 刘良乐 林荔军[2] 李奇[2] Wan Junming;Zhu Shuang;Xie Xiaobo;Lin Zhaowei;Liu Liangle;Lin Lijun;Li Qi(Department of Orthopaedics,Tongde Hospital of Zhejiang Province,Hangzhou 310002,China;Department of Orthopaedics,Zhujiang Hospital,Southern Medical University,Guangzhou 510515,China;Department of Orthopaedics,the Third Affiliated Hospital of Wenzhou Medical University,Wenzhou 325200,China)

机构地区：[1]浙江省立同德医院骨科,杭州310002 [2]南方医科大学珠江医院骨科中心,广州510515 [3]温州医科大学附属第三医院骨科,325200

出　　处：《中华骨科杂志》2020年第21期1478-1485,共8页Chinese Journal of Orthopaedics

基　　金：浙江省自然科学基金项目(LQ20H170001)。

摘　　要：目的探讨巯基聚乙二醇(polyethylene glycol-thiol,PEG-SH)修饰的GNPs/miR-29纳米微粒诱导神经干细胞增殖、分化的作用。方法采用氧化还原法制备PEG-SH修饰的GNPs/miR-29纳米微粒,检测紫外吸收光谱、粒径分布及Zeta电位。选取15只成年雄性SD大鼠,以改良Allen法构建脊髓损伤模型;在脊髓损伤区域分别植入人工合成的miR-29和PEG-SH GNPs/miR-29纳米微粒,采用凝胶电泳法分析miR-29表达的稳定性。取SPF级SD乳鼠10只,分离培养神经干细胞,使用Nestin、GFAP及NSE抗体鉴定细胞。以CCK-8法检测人工合成的miR-29、PEG-SH GNPs及PEG-SH GNPs/miR-29纳米微粒对神经干细胞活性、增殖的影响。将人工合成的miR-29、PEG-SH GNPs及PEG-SH GNPs/miR-29纳米微粒与神经干细胞共培养1周,观察神经元突起的密度、长度及数量。结果PEG-SH修饰的纳米金呈棕红色;透射电镜下为均匀分布的球形;紫外吸收光谱呈现单峰波,在523 nm附近出现吸收峰值;Zeta电位随PEG-SH含量增加而逐渐增大,峰值为(22.5±5.2)mV;粒径随PEG-SH含量增加早期迅速达峰值后下降维持至稳定水平。脊髓损伤区域植入miR-29及PEG-SH GNPs/miR-29纳米微粒0~6 h,人工合成的miR-29组可见清晰的表达条带,但12~24 h表达条带迅速消失;PEG-SH GNPs/miR-29微粒组,始终能观察到清晰的表达条带。接种细胞第1、3、5、7天,miR-29组OD值分别为0.34±0.17、0.78±0.31、1.28±0.68、1.64±0.38,与DMEM组相比差异无统计学意义;GNPs组、PEG-SH GNPs组以及PEG-SH GNPs/miR-29组的OD值与DMEM组相比差异均无统计学意义。PEG-SH GNPs/miR-29纳米微粒组神经元突起的密度为(56.38±3.65)μm2、长度为(78.25±3.72)μm、数量为(356±34.52)个/1000倍视野,均大于miR-29组[分别为(12.53±3.26)μm2、(11.35±3.36)μm、(158±32.85)个/1000倍视野]、PEG-SH GNPs组[分别为(14.12±3.45)μm2、(12.56±3.57)μm、(160±32.52)个/1000倍视野]和生理盐水组[分别为(10.25±3.52)μm2、(9.35±3.28)�Objective To prepare PEG-SH modified GNPs/miR-29 nanoparticles and to investigate the proliferation and differentiation of neural stem cells induced by PEG-SH modified GNPs/miR-29 nanoparticles.Methods PEG-SH modified GNPs/miR-29 nanoparticles were developed by oxidation-reduction method and were tested for UV absorption spectrum,particle size distribution and zeta potential of nanoparticles.A total of 15 adult male Sprague Dawley(SD)rats were used to establish spinal cord injury model by modified Allen method.The artificial miR-29 and PEG-SH modified GNPs/miR-29 nanoparticles were implanted into the injury site of spinal cord respectively.The stability of miR-29 expression was analyzed by gel electrophoresis.The neural stem cells were isolated and cultured from 10 SPF grade neonatal rats.It was identified by Nestin,GFAP and NSE antibodies.The activity and proliferation of neural stem cells in synthetic miR-29,PEG-SH GNPs and PEG-SH GNPs/miR-29 nanoparticles group was detected by CCK-8 assay.Neural stem cells were cultured with synthetic miR-29,PEG-SH GNPs and PEG-SH GNPs/miR-29 nanoparticles for 1 week.The density,length and number of neuritis were investigated.Results The solution of PEG-SH modified GNPs showed a brownish red appearance.The spheres were in uniform distribution under transmission electron microscope.The results of UV absorption spectrum showed a single peak wave.The peak value of UV absorption was near 523 nm.The zeta potential increased gradually with the increased content of PEG-SH.The peak value of zeta potential was 22.5±5.2 mV.With the increase of content of PEG,the particle size of PEG-SH modified GNPs rapidly reached peak value at the early stage and then decreased rapidly to a relatively stable level.The synthetic miR-29 and PEG-SH modified GNPs/miR-29 nanoparticles were implanted into the injury site of spinal cord.At 0-6 h,clear band was observed in the synthetic miR-29 group.However,the band was disappeared rapidly at 12-24 h.In PEG-SH GNPs/miR-29 group,clear band were always observe

关 键 词：肽类 迟效制剂 支架 干细胞 微RNAS 

分 类 号：R318.08[医药卫生—生物医学工程]