Establishment and characterization of conditionally immortalized organ of Corti cell lines
Establishment and characterization of conditionally immortalized organ of Corti cell lines
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DOI：
10.1006/cbir.1998.0339
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发表时间：
1999-01-01
影响因子：
3.9
通讯作者：
Kachar, B
中科院分区：
文献类型：
--
作者：
Kalinec, F;Kalinec, G;Kachar, B
从2周龄H-2Kb-tsA58 （Immortomouse）转基因小鼠的Corti器官中分离培养细胞。这些小鼠的所有细胞都含有类人猿病毒40a基因的突变体，该基因编码一种耐热大t抗原（Tag）蛋白。在33摄氏度时，Tag蛋白具有功能并诱导细胞增殖，但在39摄氏度时，它迅速变性和失活。在33℃下生长的Corti细胞离体器官以小、圆或梭形为主，增殖迅速。当移至39℃时，细胞增殖速率降低，分化为特定的形态表型；用极限稀释法克隆了4株细胞系，并用免疫荧光显微镜和Western blot对其进行了鉴定。命名为OC-k1， OC-k2， OC-k3和OC-k4的细胞系已传代至少50次，并保持稳定的表型。这些细胞系神经上皮前体细胞标记物nestin和内耳细胞标记物OCP2均呈阳性。此外，这些细胞对上皮细胞和神经元细胞标记物表现出反应性，但每个克隆的蛋白质表达模式不同，在33℃或39℃下生长的同一克隆细胞之间也不同。一些克隆表现出不对称的细胞分裂，这是干细胞通常具有的特征。这些细胞系有利于研究哺乳动物内耳细胞分化和形态发生控制的机制和信号，以及分离内耳特异性蛋白。(C) 1999学术出版社。
A culture of cells was isolated from the organ of Corti of 2-week-old H-2Kb-tsA58 (Immortomouse) transgenic mice. All cells of these mice harbor a mutant of the simian virus 40 A-gene, encoding a thermolabile large T-antigen (Tag) protein. At 33 degrees C the Tag protein is functional and induces cell proliferation, but at 39 degrees C it is rapidly denatured and inactivated. Isolated organ of Corti cells growing at 33 degrees C were predominantly small, rounded or fusiform and proliferated rapidly. When moved to 39 degrees C, the cells reduced their rate of proliferation and differentiated into specific morphological phenotypes; Four cell lines were cloned by limiting dilution and characterized by immunofluorescence microscopy and Western blot. The cell lines, named OC-k1, OC-k2, OC-k3 and OC-k4, have been passaged at least 50 times with retention of a stable phenotype. These cell lines were all positive for the neuroepithelial precursor cell marker nestin and for the inner ear cell marker OCP2. In addition, the cells showed reactivity to epithelial and neuronal cell markers, but with a pattern of protein expression different for each clone and different between cells of the same clone growing at 33 degrees C or 39 degrees C. Some of the clones exhibited asymmetric cell division which is a characteristic commonly ascribed to stem cells. These cell lines can be used advantageously to study mechanisms and signals involved in the control of cell differentiation and morphogenesis of the mammalian inner ear and to isolate inner ear specific proteins. (C) 1999 Academic Press.