Yang Pinglin, He Xijing, Li Haopeng, Lan Binshang, Wang Guoyu, Liu Yiheng, Li Qiang
Second Department of Orthopedics, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P.R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Feb;23(2):151-5.
To investigate the division, proliferation and differentiation abilities of nestin+/GFAP+ cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs).
Twelve male SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group in which the spinal cord injury model was established by aneurysm clip compression method, and control group in which no processing was conducted. At 5 days after modeling, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cell suspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was applied to induce differentiation. Immunohistochemistry detection and flow cytometry were applied to observe and analyze the type of cells and their capability of division, proliferation and differentiation.
At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 +/- 0.71 and 1.12 +/- 0.38, respectively, indicating there was a significant difference between two groups (P < 0.01). Concerning cell cycle, the proportion of S-phase cell and proliferation index of the model group (15.49% +/- 3.04%, 15.88% +/- 2.56%) were obviously higher than those of the control group (5.84% +/- 0.28%, 6.47% +/- 0.61%), indicating there were significant differences between two groups (P < 0.01). In the model group, primary cells gradually formed three-dimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multiple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of beta-tubulin III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ oligodendrocyte, beta-tubulin II+ neuron and GalC+ cell body and protruding were observed.
Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the ability of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
探讨脊髓损伤后巢蛋白阳性/胶质纤维酸性蛋白阳性（nestin+/GFAP+）细胞的分裂、增殖及分化能力，明确其是否具有神经干细胞（NSC）特性。
将12只8周龄、体重200 - 250 g的雄性SD大鼠随机分为2组（每组n = 6）：模型组采用动脉瘤夹压迫法建立脊髓损伤模型，对照组不做处理。建模后5天，获取每组大鼠T8脊髓节段，分离中央管周围室管膜区以外脊髓的灰质和白质制备单细胞悬液。采用无血清NSC培养基培养，血清NSC培养基诱导分化。应用免疫组织化学检测和流式细胞术观察分析细胞类型及其分裂、增殖和分化能力。
损伤后3 - 7天，模型组单细胞悬液中有大量nestin+/GFAP+细胞，而对照组很少。模型组和对照组细胞计数分别为5.15±0.71和1.12±0.38，两组间差异有统计学意义（P < 0.01）。关于细胞周期，模型组S期细胞比例和增殖指数（15.49%±3.04%，15.88%±2.56%）明显高于对照组（5.84%±0.28%，6.47%±0.61%），两组间差异有统计学意义（P < 0.01）。在模型组，原代细胞逐渐形成三维细胞克隆球，其体积小、边缘光滑、中心突出，巢蛋白免疫荧光染色呈阳性，多次传代后收获大量细胞克隆球。而在对照组，单细胞悬液的原代及传代培养均未观察到明显的细胞克隆球。模型组克隆球诱导分化5天后，免疫荧光染色显示有许多半乳糖脑苷脂（GaLC）-巢蛋白阳性细胞；5 - 7天，有大量β-微管蛋白III-巢蛋白阳性和胶质纤维酸性蛋白-巢蛋白阳性细胞；5 - 14天，观察到GaLC+少突胶质细胞、β-微管蛋白II+神经元以及GaLC+细胞体和突起。
成年大鼠脊髓压迫性损伤后，分离中央管周围室管膜区以外脊髓的灰质和白质获得的nestin+/GFAP+细胞具有自我更新能力和多向分化潜能，可能是中枢神经系统中NSC的可再生来源。
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