Truckenmiller M E, Vawter Marquis P, Zhang Peisu, Conejero-Goldberg Concha, Dillon-Carter Ora, Morales Nelly, Cheadle Chris, Becker Kevin G, Freed William J
Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Exp Neurol. 2002 Jun;175(2):318-37. doi: 10.1006/exnr.2002.7898.
Central nervous system progenitor cells that are self-renewing in culture and also differentiate under controlled conditions are potentially useful for developmental studies and for cell-based therapies. We characterized growth and plasticity properties and gene expression in a rat mesencephalic cell line, AF5, that was immortalized with an N-terminal fragment of SV40 large T (T155g). For over 150 population doublings in culture, the growth rate of AF5 cells remained steady, the cells remained responsive to bFGF, and telomerase activity and telomere lengths were unchanged. While karyotype analyses revealed some chromosomal abnormalities, these were also unchanged over time; additionally, no mutations in p53 gene sequences were found, and wild-type p53 activation was normal. AF5 cells produced PDGF, TGFbeta1, TGFbeta2, and bFGF. Similar to primary progenitor cells, AF5 cells retained their plasticity in culture; they could be propagated in an undifferentiated state as "neurospheres" in serum-free media or as adherent cultures in serum-containing media, and they differentiated when allowed to become confluent. Adherent subconfluent actively growing cultures expressed a marker for immature neurons, nestin, while few cells expressed the mature neuronal cell marker betaIII-tubulin. Confluent cultures ceased growing, developed differentiated morphologies, contained few or no nestin-expressing cells, and acquired betaIII-tubulin expression. Global gene expression was examined using a 15,000 gene microarray, comparing exponential growth with and without bFGF stimulation, and the differentiated state. The AF5 cell line exhibited stable genetic and growth properties over extended periods of time, while retaining the ability to differentiate in vitro. These data suggest that the AF5 cell line may be useful as an in vitro model system for studies of neural differentiation.
在培养中能够自我更新且在可控条件下分化的中枢神经系统祖细胞，对于发育研究和基于细胞的治疗具有潜在用途。我们对一种大鼠中脑细胞系AF5的生长、可塑性特性及基因表达进行了表征，该细胞系用SV40大T抗原的N端片段（T155g）永生化。在培养中经过超过150次群体倍增，AF5细胞的生长速率保持稳定，细胞对碱性成纤维细胞生长因子（bFGF）仍有反应，端粒酶活性和端粒长度未改变。虽然核型分析显示存在一些染色体异常，但这些异常也随时间未变；此外，未发现p53基因序列有突变，野生型p53激活正常。AF5细胞产生血小板衍生生长因子（PDGF）、转化生长因子β1（TGFβ1）、转化生长因子β2（TGFβ2）和bFGF。与原代祖细胞相似，AF5细胞在培养中保留了其可塑性；它们可以在无血清培养基中以“神经球”形式在未分化状态下增殖，或在含血清培养基中以贴壁培养形式增殖，当汇合时它们会分化。贴壁的亚汇合活跃生长培养物表达未成熟神经元的标志物巢蛋白，而很少有细胞表达成熟神经元细胞标志物βIII-微管蛋白。汇合培养物停止生长，形成分化形态，几乎没有或没有表达巢蛋白的细胞，并获得βIII-微管蛋白表达。使用15000基因微阵列检测全局基因表达，比较有无bFGF刺激时的指数生长以及分化状态。AF5细胞系在较长时间内表现出稳定的遗传和生长特性，同时保留了体外分化的能力。这些数据表明，AF5细胞系可能作为神经分化研究的体外模型系统有用。
