Conditionally immortalized cell line of inducible metanephric mesenchyme
Conditionally immortalized cell line of inducible metanephric mesenchyme
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DOI：
10.1046/j.1523-1755.2003.00010.x
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发表时间：
2003-06-01
影响因子：
19.6
通讯作者：
Perantoni, AO
中科院分区：
文献类型：
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作者：
Levashova, ZB;Plisov, SY;Perantoni, AO
背景后肾间充质（MM）在肾小管形成中的间充质-上皮转化长期以来一直是形态发生中诱导信号的范例。然而，这种分化的机制仍然是一个谜，由于原代间充质细胞的数量不足，必须手动从动物胚胎中分离。为了克服这个问题，我们已经建立了一个有条件的永生化细胞系，大鼠诱导后肾间充质（RIMM-18）通过转染的原代间充质细胞与载体，编码雌二醇依赖性E1 A-ER融合蛋白。采用逆转录-聚合酶链反应（RT-PCR）、荧光素酶报告基因分析、电泳迁移率变动分析、免疫细胞化学和免疫组化染色等方法对建立的细胞系进行鉴定。我们证明，在雌二醇的存在下，RIMM-18细胞系不断增殖，保持间充质特性超过40代。这些细胞是波形蛋白阳性和细胞角蛋白阴性。在不存在雌二醇的诱导条件下，它们响应于许多细胞因子，这些细胞因子是体内和体外间充质细胞的确定诱导剂[即，成纤维细胞生长因子2（FGF 2）、白血病抑制因子（LIF）和转化生长因子-β 2（TGF-β 2）]。我们显示在RIMM-18细胞中存在诱导MM中小管形成所需的特异性蛋白质标记物和功能活性信号传导途径。此外，诱导的RIMM-18细胞改变形态，获得上皮样特征，并且开始表达上皮标记物（例如，钙粘蛋白、细胞角蛋白、γ-谷氨酰转肽酶和分泌型卷曲相关蛋白2（sFRP 2）。RIMM-18细胞系的这一初步特征表明，它将有助于研究肾单位发育的生化和分子机制，并可能用于某些类型的肾癌，如Wilms瘤，它讽刺了肾脏发育的正常过程。
Background. The mesenchymal-epithelial conversion of metanephric mesenchyme (MM) in the formation of nephronic tubules has long served as a paradigm for inductive signaling in morphogenesis. However, the mechanisms underlying this differentiation have remained an enigma due to insufficient numbers of primary mesenchymal cells that must be isolated manually from animal embryos. To overcome this problem, we have established a conditionally immortalized cell line, the rat-inducible metanephric mesenchyme (RIMM-18) by transfection of primary mesenchymal cells with a vector, encoding an estradiol-dependent E1A-ER fusion protein.Methods. Reverse transcription-polymerase chain reaction (RT-PCR), luciferase reporter assay, electrophoretic mobility shift assay, immunocytochemical, and immunohistochemical stainings were used to characterize the established cell line.Results. We demonstrate that in the presence of estradiol, the RIMM-18 cell line proliferates continuously, maintaining mesenchymal characteristics for over 40 passages. These cells are vimentin-positive and cytokeratin-negative. Under inductive conditions in the absence of estradiol, they are responsive to a number of cytokines, which are established inducers of mesenchymal cells in vivo and in vitro [i.e., fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and transforming growth factor-beta2 (TGF-beta2)]. We show the presence in RIMM-18 cells of specific protein markers and functionally active signaling pathways required for induction of tubule formation in MM. Furthermore, induced RIMM-18 cells change morphology, acquiring epithelial-like features, and begin to express epithelial markers (e.g., E-cadherin, cytokeratin, gamma-glutamyl-transpeptidase, and secreted frizzled-related protein 2 (sFRP2).Conclusion. This preliminary characterization of the RIMM-18cell line suggests that it will be useful in the study of biochemical and molecular mechanisms of nephronic development and, possibly, of some types of renal cancer such as Wilms' tumor, which caricatures the normal process of kidney development.