In vitro culture and differentiationof goat neural stem cells

doi:10.3969/j.issn.2095-4344.2014.23.014

Abstract

Abstract:

BACKGROUND: Different types of nerve regulatory factors and glial cells have been reported to exert different roles in the differentiation and maturation of neural stem cells, but culturing neural stem cells in large animals is relatively rarely reported.

OBJECTIVE: To explore the way for culturing goat neural stem cells and to detect the outcome after in vitro differentiation.

METHODS: The neural stem cell was separated and cultured from the newborn goat cerebral cortex and the anti-nestin immunocytochemical staining was performed for cell identification. At the same time, anti-S100 active Schwann cells were gotten from the sciatic nerve. Then in vitro differentiation was preformed and the outcome was detected by the immunocytochemical stain of anti-glial fibrillary acidic protein, anti-microtubule-associated protein 2 and anti-S100. Cells without primary antibodies served as controls. Gray values were calculated and compared.

RESULTS AND CONCLUSION: The Schwann cells were cultured successfully, which were active to the anti-nestin immunocytochemical staining and anti-S100 staining. After differentiation, the products were active to anti-glial fibrillary acidic protein and anti-microtubule-associated protein 2 immunocytochemical stain, but

negative to the anti-S100. And significant difference was found in gray values. The goat neural stem cells and Schwann cells were successfully cultured and identified. After the differentiation, the astrocytes and neurons were detected, but the Schwann cells were not found.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, neural stem cells, cells, cultured, goats

CLC Number:

R394.2

Xia Xu-ke1, 2, Zhang Wei3, Yu Jie-feng1, 4, Zhang Ying1. In vitro culture and differentiationof goat neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3691-3695.

Figures/Tables 2

2.1 神经干细胞培养及抗-巢蛋白免疫组织化学鉴定细胞悬液培养5 d后于倒置相差显微镜下观察，可见强折光性神经球悬浮生长于培养液内，边界清晰，由2-4个细胞组成，并可见少量组织碎块及部分死细胞漂浮于培养液中，肉眼观培养液呈橙黄色，微浊。换液2次后组织碎块及死细胞数量减少，神经球明显增大(图1A)。传代后再次于镜下观察，可见细胞团呈强折光性，直径较前减小。第3代神经球表达神经干细胞特异性巢蛋白，行抗-巢蛋白免疫组织化学检测呈阳性反应(图1B)。 2.2 许旺细胞纯化培养及抗-S100免疫组织化学鉴定组织块接种培养三四天后培养液呈淡黄色，可见大量坏死组织块漂浮于液面，此时倾出培养液及坏死组织，可见少量组织块贴附于培养瓶内，镜下观察可见自附壁组织块周缘爬行生长出条索状细胞若干，细胞两端尖细，中部膨隆，可见胞核。细胞自附壁组织块周缘向外呈放射性生长。另可见培养瓶内散在分布不规则形状细胞，包体不规则向外突起，核大，居中。传代培养并利用前述差速贴壁法纯化后可见瓶内均匀分布大量条索状细胞，细胞排列较紧密，具有“肩并肩、手拉手”样特征。抗-S100免疫荧光染色后于荧光显微镜下可见细胞核及胞体发出黄绿色荧光(图1C)。 2.3 神经干细胞诱导分化及抗-胶质纤维酸性蛋白/抗- S100/抗-MAP2免疫组织化学鉴定以诱导分化培养液培养7 d后于倒置相差显微镜下观察，可见大量神经球贴壁，自外周发出突触若干，突触细长，具有分支。部分细胞形态改变，胞体变大，呈圆形或多角形，并向四周发出突触。抗-胶质纤维酸性蛋白及抗-MAP2免疫荧光检测见大量细胞核、包体及突触发出绿色荧光(图2A，B)，DAPI复染后可见胞核发出蓝色荧光(图2C)，抗-S100免疫荧光未检见阳性产物。 2.4 图像灰度量化及统计学比较结果按前所述以ImageJ1.47图像分析软件测量各组图像灰度值，并以SPSS13.0统计分析软件对结果进行统计学比较(表1)。结果显示，经神经干细胞体外分化后的细胞抗-巢蛋白、抗-MAP2、抗胶质纤维酸性蛋白免疫组织化学染色各阳性组与对应阴性对照组灰度值均数比较差异均有显著性意义(P < 0.001)；分化后细胞行抗-S100免疫组织化学染色阴性组与阳性对照组(许旺细胞进行抗-S100免疫组织化学染色)灰度值均数进行比较差异有显著性意义(P < 0.001)。"

References

[1] Noctor SC, Martínez-Cerdeño V, Ivic L,et al.Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.Nat Neurosci.2004.7(2): 136-144.
[2] 刘伟国,虞军,龚江标.大鼠胚胎神经干细胞的培养及分化的观察[J].江医学,2005,27(1):30-32.
[3] 曹李,张宇新.神经干细胞移植治疗的研究进展[J].人民军医，2012,55(3):266-267.
[4] Cui L, Jiang J.Transplantation of embryonic stem cells improves nerve repair and functional recovery after severe sciatic nerve axotomy in rats.Stem Cells. 2008; 26(5): 1356-1365.
[5] Xu L, Zhou S, Feng GY,et al. Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats. Mol Neurobiol.2012;46(2):265-274.
[6] Roy NS, Cleren C, Singh SK, et al.Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med.2006;12(11): 1259-1268.
[7] Gage FH. Mammalian neural stem cells. Science. 2000; 287(5457): 1433-1438.
[8] Chojnacki A, Weiss S.Production of neurons, astrocytes and oligodendrocytes from mammalian CNS stem cells. Nat Protoc. 2008;3(6):935-940.
[9] Tansey KE, Seifert JL, Botterman B, et al.Peripheral Nerve Repair Through Multi-Luminal Biosynthetic Implants.Ann Biomed Eng.2011;39(6):1815-1828.
[10] Liard O, Segura S, Sagui E,et al. Adult-brain-derived neural stem cells grafting into a vein bridge increases postlesional recovery and regeneration in a peripheral nerve of adult pig.Stem Cells Int. 2012;2012:128732.
[11] 李峰,穆广态.外周神经损伤的显微外科修复[J].中华显微外科杂志,2004, 27(1):27-29.
[12] 蔡明,陆耀飞,娄淑杰.大鼠大脑皮层神经干细胞培养方法比较[J].神经解剖学杂志,2013,29(1):70-74.
[13] Riet ze RL, Valcanis H, Brooker GF, et al. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature. 2001;412(6848) : 736-739.
[14] 王璐,何芙蓉.巢蛋白nestin在人甲状腺常见肿瘤组织的表达[J].实用肿瘤杂志,2010. 25(5):515-517.
[15] 张俊虎,王建交.神经干细胞的鉴定[J].中国组织工程研究,2012, 16(27):108-5112.
[16] 张在金, 董艳, 卢亦成.大鼠胎脑不同部位神经干细胞比较性培养的研究[J].中华神经外科疾病研究杂志, 2008, 7(4):325-327.
[17] 刘靖,曲静,徐晓静,等.神经干细胞迁移的研究进展[J].中国细胞生物学学报.2012,34(3):201-211.
[18] 罗江兵,赵鹏洲,欧英雄,等.神经干细胞在脑损伤模型中的迁移、成活和神经生长因子表达[J].中国微侵袭神经外科杂志,2012, 17(5):232-234.
[19] 刘岚,张成.大鼠脂肪干细胞源性神经球分化为雪旺细胞样细胞[J].南京医科大学学报:自然科学版,2011,31(7):991-995.
[20] 万虹,安沂华.雪旺氏细胞促进共培养大鼠胚胎神经干细胞的分化[J].中华神经外科杂志,2002,18(2):100-103.