大鼠原代背根神经元和雪旺细胞在随机及有序排列聚甲基丙烯酸甲酯电纺纳米纤维上共培养的形态学

Morphology of rat dorsal root ganglion neurons and Schwann cells cocultured on polymethylmethacrylate electrospun nanofibers with different topographic features

目的:探讨聚甲基丙烯酸甲酯(PMMA)电纺纳米纤维支架的拓扑线索对于大鼠原代背根神经元(DRGn)培养及其与雪旺细胞(SCs)共培养的影响。方法:构建具有随机分布和轴向有序排列拓扑结构的PMMA电纺纳米纤维,分别为随机和有序PMMA电纺纳米纤维组,以PMMA薄膜作为对照组;分离纯化大鼠原代DRGn和SCs,与上述各组PMMA电纺纳米纤维共培养;利用慢病毒技术转染荧光蛋白基因作为显色方法,观察PMMA电纺纳米纤维的拓扑线索对于DRGn神经突生长的影响,在共培养实验通过荧光图像的快速傅立叶转换(FFT)及半高全宽值(FWHM)的计算,定量分析电纺纤维对DRGn神经突和SCs细胞突起的接触引导作用。结果:大鼠原代DRGn和SCs均能够顺利在PMMA材料上贴壁并生长;与PMMA薄膜组比较,随机和有序PMMA电纺纳米纤维组DRGn平均神经突数量及神经突长度差异无统计学意义(P>0.05);共培养实验中,电纺纤维的拓扑线索对于DRGn神经突和SCs细胞突起的生长均具有明显的接触引导作用,与PMMA薄膜组和随机PMMA电纺纳米纤维组比较,有序PMMA电纺纳米纤维组DRGn和SCs的FWHM值均明显降低(P<0.01),有序PMMA电纺纳米纤维能够在空间上促成DRGn神经突和SCs细胞突起建立共定位。结论:有序PMMA电纺纳米纤维具有作为脊髓损伤(SCI)后植入性支架材料的潜力,其拓扑线索有可能加速SCs的轴突髓鞘化过程。

Objective.. To evaluate the influence of the topographic features of polymethylmethacrylate （PMMA） electrospun nanofibers in the culture of rat dorsal root ganglion neurons （DRGn） and its coculture with Sehwann cells （SCs）. Methods： The random and aligned electrospun PMMA nanofibers were constructed to culture the rat DRGn or coculture it with SCs. PMMA film was used as control group. The lentivirus transfected fluorescence reporting gene was used as the coloring method, and the influence of the pographic features of PMMA electrospun nanofibers in the growth of DRGn was observed; in coculture experiment, Fast Fourier Transform of the fluorescence images and Full-width/Half maximum were calculated, and the effects of electrospun fibers on the DRGn and SCs were analyzed. Re.suits： The DRGn and SCs were shown capable to adhere to and grow on PMMA nanofibers respectively; compared with PMMA film group, the average neurite number and average neurite lengthes of the DRGn in radnom and aligned PMMA electrospun nanofibers groups had no significant differences （P〉0.05）. In coculture experiment, PMMA electrospun nanofibers could exert obvious contact guidance on both neurite growth of DRGn and cell process growth of SCs; compared with the cells on PMMA film or random nanofibers, the FWHM of DRGn and SCs on aligned PMMA nanofibers was significantly decreased, and the aligned PMMA nanofibers could promote the neurites of DRGn and SCs to form colocalization. Conclusion： Aligned PMMA electrospun nanofibers have the potentiality as transplantable scaffold after spinal cord injury （SCI）, and the topographic features may advance the process of axon myelination of SCs.