Cai Wenyi, Jiang Yuting, Xu Xuelian, Ma Qiliang, Xu Congcong, Fu Wei, Lan Daoliang
College of Animal & Verterinary Sciences, Southwest Minzu University, Chengdu 610041, China.
Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education, Southwest Minzu University, Chengdu 610041, China.
Animals (Basel). 2025 Aug 26;15(17):2509. doi: 10.3390/ani15172509.
Oviduct epithelial cells (OECs) constitute a critical component of the oviductal mucosa, providing essential microenvironmental support for fertilization and early embryonic development. Their frequent application in embryo co-culture systems is constrained in yaks () by limited tissue availability and the short lifespan of primary yak oviduct epithelial cells (YOECs). To address this limitation, we established immortalized YOEC lines using a lentiviral vector system. Primary YOECs isolated from reproductive tract tissues of adult female yaks via enzymatic digestion were immortalized through individual and combined transfection with simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). The resulting immortalized lines (YOECs-S: SV40LT alone; YOECs-HS: dual SV40LT/hTERT) kept their typical cobblestone shape and still made cytokeratin 18. Both lines exhibited stable SV40LT and hTERT expression ( > 50), maintained diploid karyotypes, and demonstrated serum-dependent growth, contact inhibition, and hormone responsiveness. Notably, YOECs-HS displayed superior proliferative capacity and phenotypic stability during long-term culture. This study reports the first successful establishment and comprehensive characterization of immortalized YOEC lines. These validated models provide a valuable experimental platform for optimizing yak embryo-oviduct epithelial cell co-culture systems and advancing reproductive research in this high-altitude-adapted species.
输卵管上皮细胞（OECs）是输卵管黏膜的关键组成部分，为受精和早期胚胎发育提供重要的微环境支持。在牦牛中，由于组织获取有限以及原代牦牛输卵管上皮细胞（YOECs）寿命较短，它们在胚胎共培养系统中的频繁应用受到限制。为解决这一限制，我们使用慢病毒载体系统建立了永生化的YOEC系。通过酶消化从成年雌性牦牛生殖道组织中分离出的原代YOECs，经单独或与猿猴病毒40大T抗原（SV40LT）和人端粒酶逆转录酶（hTERT）联合转染实现永生化。所得的永生化细胞系（YOECs-S：仅SV40LT；YOECs-HS：SV40LT/hTERT双转染）保持其典型的鹅卵石形状，仍能产生细胞角蛋白18。两个细胞系均表现出稳定的SV40LT和hTERT表达（>50），维持二倍体核型，并表现出血清依赖性生长、接触抑制和激素反应性。值得注意的是，YOECs-HS在长期培养过程中表现出更强的增殖能力和表型稳定性。本研究首次成功建立并全面表征了永生化的YOEC系。这些经过验证的模型为优化牦牛胚胎 - 输卵管上皮细胞共培养系统以及推进这种适应高海拔物种的生殖研究提供了宝贵的实验平台。
