Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.
Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.
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DOI：
10.1371/journal.pone.0095919
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Kültz D
中科院分区：
文献类型：
--
作者：
Gardell AM;Qin Q;Rice RH;Li J;Kültz D
鱼类细胞培养正成为研究对环境挑战的生理反应的分子机制的越来越广泛的模型。在这项研究中，我们从脑(OMB)和嘴唇上皮(OML)中获得了两个永生化的莫桑比克罗非鱼(Oreochromis Mossambius)细胞系，并将它们与先前永生化的动脉球部(TMB)细胞系进行了比较。OMB和OML细胞系在没有或有Rho相关激酶(ROCK)抑制剂/3T3饲养层补充的情况下产生。虽然这两种方法都是成功的，但补充ROCK抑制剂/饲养层被发现提供了选择上皮样细胞类型和缩短永生化时间的优势。在永生化(≥第5代)后，我们用激光共聚焦显微镜对新衍生的细胞系(OMB和OML)的蛋白质组进行了鉴定，并为每一株鉴定了几个独特的细胞标记。随后，评估了三种细胞系在急性暴露于高浓度氯化钠后的渗透耐受性。这些罗非鱼细胞系的急性最大渗透耐受性(>；700mOsm/kg)显著高于任何已知的脊椎动物细胞系，但在上皮样OML细胞系中显著高于其他脊椎动物细胞系。为了验证这些罗非鱼细胞系的生理相关性，我们量化了急性高渗透攻击(450mOsm/kg和700mOsm/kg)对两种酶的转录调控的影响，这两种酶参与了相容的有机渗透调节物质肌醇的生物合成。这两种酶在所有三个罗非鱼细胞系中都被发现强烈上调。因此，新建立的罗非鱼细胞系是研究泛盐目鱼类渗透胁迫反应的分子机制的有价值的工具。
Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.
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