成年大鼠压迫性脊髓损伤后损伤区神经干细胞的分离培养实验研究

ISOLATION AND CULTURE OF NEURAL STEM CELLS IN INJURED REGION OF COMPRESSIVE SPINAL CORD INJURY IN ADULT RAT

目的研究脊髓损伤后损伤反应性巢蛋白(nestin)和胶质酸性纤维蛋白(glial fi brillary acid protein,GFAP)阳性共存(nestin+/GFAP+)细胞的分裂、增殖和分化能力,以探讨其是否具有神经干细胞(neural stem cells,NSCs)特性。方法8周龄雄性SD大鼠12只,体重200～250 g,随机分为正常对照组和模型组(n=6)。模型组利用动脉瘤夹压迫法建立成年大鼠脊髓损伤动物模型,正常对照组不作任何处理。造模后5 d,两组分别取大鼠T8脊髓节段,分离中央管周围室管膜区以外的脊髓灰质和白质,制成单细胞悬液,用无血清NSCs培养基进行培养,并用含血清NSCs培养基进行诱导分化,利用免疫荧光化学和流式细胞仪观察细胞类型及分裂、分化、增殖能力。结果模型组培养后3～7 d,单细胞悬液中有大量高度表达的nestin+/GFAP+共存细胞,细胞计数为5.15±0.71;对照组为1.12±0.38;两组比较差异有统计学意义(P<0.01)。细胞周期结果示,模型组S期细胞比例(15.49%±3.04%)及增殖指数(15.88%±2.56%)均明显高于对照组(5.84%±0.28%,6.47%±0.61%),两组比较差异有统计学意义(P<0.01)。模型组原代细胞逐渐形成边缘光滑、中心膨隆有立体感的小克隆球,nestin免疫荧光染色呈强阳性,多次传代后获得大量细胞克隆球。对照组单细胞悬液原代及传代培养均未见明显克隆球生长。免疫染色结果示模型组克隆球诱导分化约5 d,细胞球中含有大量半乳糖脑苷脂(galactocerebroside,GaLC)-nestin免疫染色阳性细胞;5～7 d,大量β-微管蛋白Ⅲ(β-tubulinⅢ)-nestin和GFAP免疫染色阳性细胞;7～14 d出现GaLC阳性少突胶质细胞、β-tubulinⅢ神经元和GFAP染色阳性的胞体及细胞突起。结论成年大鼠压迫性脊髓损伤后,剔除中央管周围室管膜区脊髓白质与灰质分离而得的nestin+/GFAP+细胞,具有自我更新能力和多分化潜能,是中枢神经系统的NSCs。

Objective 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）. Methods 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, Ta 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. Results 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 threedimensional 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 β-tubulin Ⅲ-nestin＋ and GFAP-nestin＋ cells; and at 5-14 days, GaLC＋ oligodendrocyte, ]3-tubulin Ⅱ＋ neuron and Gal C＋ cell body and protruding were observed, Conclusion 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.