Establishment and characterization of neonatal mouse sertoli cell lines.
Establishment and characterization of neonatal mouse sertoli cell lines.
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DOI：
10.1002/j.1939-4640.2003.tb02650.x
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发表时间：
2003-01
影响因子：
--
通讯作者：
M. Hofmann;K. S. Van Der Wee;Jamie L. Dargart;G. Dirami;L. Dettin;M. Dym
中科院分区：
文献类型：
--
作者：
M. Hofmann;K. S. Van Der Wee;Jamie L. Dargart;G. Dirami;L. Dettin;M. Dym
用猴病毒40大肿瘤抗原基因(SV40-LTAg)使产后6天小鼠睾丸支持细胞在蜕皮激素类似物--蜕皮激素A可诱导的启动子的控制下条件永生化。这一策略产生了2个细胞系，它们表现出混合的表型。我们首先测试了LTAg基因的条件性表达，结果表明，当诱导剂存在于培养基中时，两株细胞都表达了LTAg。当去掉Ponasterone A时，大多数细胞死亡。然而，经过60代后，在没有激素的情况下LTAg的持续表达表明，细胞的基因组可能发生了未知的变化。进一步亚克隆其中一个细胞系，获得了7个新的细胞系，其形态与组织培养中的Sertoli细胞相似。对每个细胞系收集的RNA进行逆转录聚合酶链式反应(RT-PCR)，以确定哪些细胞与体内Sertoli细胞表型相似。除α-抑制素、GATA-1和类固醇生成因子-1外，所有细胞株均表达支持细胞特异性基因干细胞因子(SCF)和硫酸糖蛋白-2(SGP-2)的产物。此外，这些细胞株表达已知的在体内和体外作用于生殖细胞的生长和分化因子，如白血病抑制因子(LIF)、转化生长因子β(TGF-β)和碱性成纤维细胞生长因子(BFGF)。此外，当用作共培养的饲养层时，这些品系中至少有2个能够保持A型精原细胞的活力至少7天，并支持精原细胞分化的第一步。
Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation.