Isolation and characterization of mouse neural precursor cells in primary culture
Isolation and characterization of mouse neural precursor cells in primary culture
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原代培养中小鼠神经前体细胞的分离和表征
DOI：
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发表时间：
1991
期刊：
影响因子：
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通讯作者：
Y. Tomooka
中科院分区：
文献类型：
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作者：
H. Kitani;R. Shiurba;T. Sakakura;Y. Tomooka
小鼠神经前体细胞的原代培养是通过酶促分离胚胎第10天胎头，然后阴性选择非神经污染细胞来建立的。在允许神经前体细胞保持悬浮在培养基中的条件下，后者被允许附着并散布在塑料基底上。然后将所得神经上皮细胞富集的悬浮液铺在用聚-d-赖氨酸包被的培养皿上。成纤维细胞的生长在选择性培养基中受到抑制。细胞增殖通过溴脱氧尿苷标记后的细胞核的免疫过氧化物酶染色来测量。标记细胞的比例下降，从50%，第1天，直到第5天，当它接近零，并在培养7天后，在培养基中含有1%的胎牛血清，转铁蛋白，胰岛素，霍乱毒素，亚硒酸钠的细胞数增加了四倍。神经前体细胞的分化进行了研究，通过间接免疫荧光显微镜的神经元和星形胶质细胞特异性的细胞骨架蛋白的外观在连续的时间间隔在文化。在第1天，携带神经炎突起并表达神经丝以及微管相关蛋白2的细胞数量较少，直至第14天增加。星形胶质细胞的形态特征和胶质细胞酸性蛋白的免疫反应性的星状细胞，直到第5天才被检测到，并没有成为丰富的，直到第11天。在形态学或免疫细胞化学染色特征没有差异，发现神经前体细胞的酶促解离处理的整个胎儿头和那些恢复胎儿神经上皮细胞的手工解剖。通过这种快速，简单的方法获得大量的神经前体细胞，使得大量培养物的生产成为可能，用于对小鼠中枢神经系统早期发育过程中控制增殖和分化的调节因子进行分子分析。
SummaryPrimary cultures of mouse neural precursor cells were established by enzymatic dissociation of embryonic Day 10 fetal heads followed by negative selection of non-neural contaminating cells. The latter were allowed to attach and spread on a plastic substrate under conditions that permitted neural precursor cells to remain suspended in the culture medium. The resulting neuroepithelial cell enriched suspension then was plated on dishes coated with poly-d-lysine. Growth of fibroblastic cells was inhibited in a selective medium. Cell proliferation was measured by immunoperoxidase staining of nuclei after bromodeoxyuridine labeling. The proportion of labeled cells declined from 50% on Day 1 until Day 5 when it approached zero, and after 7 days in culture a fourfold increase in cell number was achieved in medium containing 1% fetal bovine serum, transferrin, insulin, cholera toxin, and sodium selenite. Differentiation of neural precursor cells was studied by indirect immunofluorescence microscopy for the appearance of neuron- and astrocyte-specific cytoskeletal proteins at successive intervals in culture. Cells bearing neuritic processes and expressing neurofilaments as well as microtubule-associated protein 2 were present in low numbers on Day 1, increasing through Day 14. Stellate cells with morphologic features of astrocytes and immunoreactive for glial fibrillary acidic protein were not detected until Day 5 and did not become abundant until Day 11. No differences in morphology or immunocytochemical staining characteristics were found between neural precursor cells processed by enzymatic dissociation of whole fetal heads and those recovered by manual dissection of fetal neuroepithelia. The large number of neural precursor cells obtained by this rapid, simple method makes possible the production of mass cultures for molecular analysis of the regulatory factors that control proliferation and differentiation during early development of the mouse central nervous system.