Primary Cell Culture Systems for Human Thyroid Studies.
Primary Cell Culture Systems for Human Thyroid Studies.
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DOI：
10.1089/thy.2015.0518
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发表时间：
2016-08
期刊：
影响因子：
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通讯作者：
Yanqiang Wang;Wei Li-;J. Phay;Rulong Z. Shen;N. Pellegata;M. Saji;M. Ringel;A. de la Chapelle;Huiling He
中科院分区：
文献类型：
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作者：
Yanqiang Wang;Wei Li-;J. Phay;Rulong Z. Shen;N. Pellegata;M. Saji;M. Ringel;A. de la Chapelle;Huiling He
背景细胞模型是甲状腺体外研究的重要工具。在实验室研究中广泛使用的永久性甲状腺细胞系通常来自肿瘤。出于许多目的，希望将肿瘤细胞与来自正常组织的细胞进行比较。然而，这些培养物生长缓慢，寿命非常有限，而且众所周知会失去它们的甲状腺特征。本研究的目的是在不同的培养系统中对编码和非编码的甲状腺标志物进行分型，以确定体外实验的最佳条件。方法从组织学非肿瘤性组织中分离人原代甲状腺细胞。使用两种可供选择的培养液(6H和h7H)。随后的单层(二维)培养的形态和行为通过显微镜进行监测。分别于免疫后第8、21、43天用逆转录聚合酶链式反应检测关键甲状腺相关基因的表达。作为先导研究，在三维吊滴培养系统中对相同的标记进行了研究。结果在6H和H7H培养液中，原代甲状腺细胞在数量和大小上均有生长。在培养的前两周，大多数细胞保持了甲状腺细胞的主要形态特征，并在第19天左右出现成纤维细胞样细胞。第21天，大多数甲状腺基因标记被保留，但到第43天，一些标记不再存在。LncRNA转录本PTCSC2(剪接)和PTCSC3最先消失。在文化的早期阶段，这两种媒体并没有根本的区别。在三维系统中，大多数甲状腺标志物在第21天前被保留。结论甲状腺细胞培养至第21天仍具有多种甲状腺特性。此后，成纤维细胞样脱分化细胞开始占优势。
BACKGROUND Cell models are key instruments for in vitro studies of the thyroid. Permanent thyroid cell lines that are widely used in laboratory research typically originate from tumors. For many purposes, it is desirable to compare tumor cells with cells originating from normal tissue. However, such cultures grow slowly, have a highly limited life-span, and are known to lose their thyroid characteristics. The aim of the present study was to type coding and noncoding thyroid markers in different culture systems in an attempt to determine the optimal conditions for in vitro experimentation. METHODS Human primary thyroid cells were isolated from histologically non-tumorous tissues. Two alternative media (6H and h7H) were used. The morphology and behavior of the ensuing monolayer (two-dimensional) cultures was monitored by microscopy. The expression of key thyroid-related genes (n = 9) was monitored by reverse transcription polymerase chain reaction on days 8, 21, and 43 after initiation. As a pilot study, the same markers were studied in a three-dimensional hanging-drop culture system. RESULTS In the cultures with 6H or h7H medium, the primary thyroid cells displayed growth in numbers and size. Most cells retained the main morphological characteristics of thyroid cells throughout the first two weeks of culture, and fibroblast-like cells appeared around day 19. By day 21, most thyroid gene markers were retained, but by day 43, several markers were no longer present. The lncRNA transcripts PTCSC2 (spliced) and PTCSC3 were the first to disappear. There were no fundamental differences between the two media in the early period of culture. In the three-dimensional system, most thyroid markers were retained by day 21. CONCLUSION Cultures of thyroid cells retain many thyroid characteristics up to day 21. Thereafter, fibroblast-like dedifferentiated cells begin to dominate.