The blood-testis barrier (BTB) formed by adjacent Sertoli cells (SCs) limits the entry of many chemicals into seminiferous tubules. Differences in rodent and human substrate-transporter selectivity or kinetics can misrepresent conclusions drawn using rodent in vitro models. Therefore, human in vitro models are preferable when studying transporter dynamics at the BTB. This study describes a hTERT-immortalized human SC line (hT-SerC) with significantly increased replication capacity and minor phenotypic alterations compared to primary human SCs. Notably, hT-SerCs retained similar morphology and minimal changes to mRNA expression of several common SC genes, including AR and FSHR. The mRNA expression of most xenobiotic transporters was within the 2-fold difference threshold in RT-qPCR analysis with some exceptions (OAT3, OCT3, OCTN1, OATP3A1, OATP4A1, ENT1, and ENT2). Functional analysis of the equilibrative nucleoside transporters (ENTs) revealed that primary human SCs and hT-SerCs predominantly express ENT1 with minimal ENT2 expression at the plasma membrane. ENT1-mediated uptake of [³H] uridine was linear over 10 min and inhibited by NBMPR with an IC₅₀ value of 1.35 ± 0.37 nM. These results demonstrate that hT-SerCs can functionally model elements of transport across the human BTB, potentially leading to identification of other transport pathways for xenobiotics, and will guide drug discovery efforts in developing effective BTB-permeable compounds.

中文翻译：

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生成 hTERT 永生化人类支持细胞模型以研究血液睾丸屏障处的转运蛋白动力学

由相邻的支持细胞 (SCs) 形成的血睾丸屏障 (BTB) 限制了许多化学物质进入曲细精管。啮齿动物和人类底物转运蛋白选择性或动力学的差异可能会歪曲使用啮齿动物体外模型得出的结论。因此，在 BTB 研究转运蛋白动力学时，人体体外模型更可取。本研究描述了一种 hTERT 永生化人类 SC 系 (hT-SerC)，与原代人类 SC 相比，其复制能力显着提高，表型变化较小。值得注意的是，hT-SerCs 保留了相似的形态，并且对几种常见 SC 基因（包括 AR 和 FSHR）的 mRNA 表达变化最小。大多数外源性转运蛋白的 mRNA 表达在 RT-qPCR 分析中的 2 倍差异阈值内，但有一些例外（OAT3、OCT3、OCTN1、OATP3A1、OATP4A1、ENT1 和 ENT2）。平衡核苷转运蛋白 (ENTs) 的功能分析表明，原代人类 SCs 和 hT-SerCs 主要表达 ENT1，而质膜上的 ENT2 表达最少。ENT1介导的[³ H] 尿苷在 10 分钟内呈线性并被 NBMPR 抑制，IC ₅₀值为 1.35 ± 0.37 nM。这些结果表明，hT-SerCs 可以在功能上模拟跨人类 BTB 的转运元素，可能导致识别异生物质的其他转运途径，并将指导药物发现工作以开发有效的 BTB 可渗透化合物。