您的账号已在其他设备登录,您当前账号已强迫下线,
如非您本人操作,建议您在会员中心进行密码修改

确定

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

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) μm 2、长度为(78.25±3.72) μm、数量为(356±34.52)个/1 000倍视野,均大于miR-29组[分别为(12.53±3.26) μm 2、(11.35±3.36) μm、(158±32.85)个/1 000倍视野]、PEG-SH GNPs组[分别为(14.12±3.45) μm 2、(12.56±3.57) μm、(160±32.52)个/1 000倍视野]和生理盐水组[分别为(10.25±3.52) μm 2、(9.35±3.28) μm、(152±32.28)个/1 000倍视野],但与胎牛血清组[分别为(56.48±3.56) μm 2、(76.85±3.65) μm、(350±35.26)个/1 000倍视野]比较差异无统计学意义。 结论:PEG-SH修饰的GNPs/miR-29纳米微粒具备良好的生物学性能,可诱导神经干细胞增殖、分化,对miR-29有一定程度的保护效应。

更多
abstracts:

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 observed. The OD values of miR-29 group were 0.34±0.17, 0.78±0.31, 1.28±0.68, 1.64±0.38 at 1, 3, 5 and 7 d after inoculation respectively. There was no significant difference in OD values compared with DMEM group. There was no significant difference in OD values among GNPs, PEG-SH GNPs, PEG-SH GNPs/miR-29 and DMEM group. The density (56.38±3.65 μm 2), length (78.25±3.72 μm) and the number [(356±34.52) /1,000×high power field] of neurites in PEG-SH GNPs/miR-29 group were higher than those in miR-29 group, PEG-SH modified GNPs group and saline group. However, there was no significant difference in the density, length and number of neurite between PEG-SH GNPs/miR-29 and serum group. Conclusion:PEG-SH modified GNPs/miR-29 nanoparticless have good biological properties. It can induce the proliferation and differentiation of neural stem cells with protective effects on miR-29.

More
作者: 万俊明 [1] 朱爽 [2] 谢小波 [2] 林昭伟 [2] 刘良乐 [3] 林荔军 [2] 李奇 [2]
作者单位: 浙江省立同德医院骨科,杭州 310002 [1] 南方医科大学珠江医院骨科中心,广州 510515 [2] 温州医科大学附属第三医院骨科 325200 [3]
期刊: 《中华骨科杂志》2020年40卷21期 1478-1485页 ISTICPKUCSCDCA
栏目名称: 基础研究
DOI: 10.3760/cma.j.cn121113-20200629-00411
发布时间: 2020-11-16
  • 浏览:8
  • 下载:2

加载中!

相似文献

  • 中文期刊
  • 外文期刊
  • 学位论文
  • 会议论文

加载中!

加载中!

加载中!

加载中!