Immortalization of porcine placental trophoblast cells through reconstitution of telomerase activity
Immortalization of porcine placental trophoblast cells through reconstitution of telomerase activity
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通过端粒酶活性重建实现猪胎盘滋养层细胞的永生化
DOI：
10.1016/j.theriogenology.2016.01.006
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发表时间：
2016-05-01
期刊：
影响因子：
2.8
通讯作者：
Tong, Dewen
中科院分区：
文献类型：
--
作者：
Zhang, Hongling;Huang, Yong;Tong, Dewen
胎盘滋养层细胞（PTCs）在组织营养性营养吸收、气体交换、内分泌活动以及母胎系统之间的屏障功能中起着关键作用。永生化猪胎盘滋养细胞的建立将有助于我们研究不同病毒对猪胎盘滋养细胞的潜在影响。在本研究中，通过胶原酶消化、梯度离心和抗CD 9免疫磁性阴性选择，从妊娠第30天至第50天的健康后备母猪中分离原代猪PTC。为了提供稳定和长寿命的细胞，原代PTC转染人端粒酶逆转录酶（hTERT）基因。选择一种命名为hTERT-PTC的猪胎盘滋养层细胞系进行表征。人端粒酶逆转录酶-PTC在体内或体外均能延长复制寿命，而不表现出任何肿瘤转化迹象。永生化PTC的形态和关键生理特征与原代PTC相似。永生化PTC保留原始细胞极性和正常核型，表达滋养层特异性标记细胞角蛋白7和E-钙粘蛋白，但不表达波形蛋白和主要组织相容性复合物I类抗原以及初级PTC。人端粒酶逆转录酶-PTCs分泌低水平的绒毛膜促性腺激素亚单位和胎盘催乳素thk与原发性PTCs一致。总之，我们的结果表明，猪PTC可以通过重建端粒酶活性永生化。永生化猪胎盘滋养细胞保持了原有的特性，可作为研究病毒感染性疾病中胎盘滋养细胞病理变化的模型细胞。(C)2016 Elsevier Inc. All rights reserved.
Placental trophoblast cells (PTCs) play a critical role in histotrophic nutrient absorption, gaseous exchange, endocrine activities, and barrier function between the maternal and fetal systems. Establishment of immortalized porcine PTCs will help us to investigate the potential effects of different viruses on porcine trophoblast. In the present study, primary porcine PTCs were isolated from healthy gilts at Day 30 to Day 50 of gestation through collagenase digestion, percoll gradient centrifugation, and anti-CD9 immunomagnetic negative selection. To provide stable and long lifespan cells, primary PTCs were transfected with human telomerase reverse transcriptase (hTERT) gene. One porcine placental trophoblast cell line, named as hTERT-PTCs, was chosen for characterization. Human telomerase reverse transcriptase-PTCs achieved an extended replicative lifespan without exhibiting any neoplastic transformation signs in vivo or in vitro. The morphologic and key physiological characteristics of the immortalized PTCs were similar to primary PTCs. The immortalized PTCs retained original cell polarity and normal karyotype, expressed trophoblast-specific marker cytokeratin 7 and E-cadherin but did not express vimentin and major histocompatibility complex class I antigens as well as primary PTCs. Human telomerase reverse transcriptase-PTCs secreted low levels of chorionic gonadotrophin subunit and placental lactogen thk were coincident with primary PTCs. Taken together, our results demonstrated that the porcine PTCs could be immortalized through reconstitution of telomerase activity. The immortalized PTCs maintained its original characteristics and can be used as a model cells line to study the pathologic changes of porcine placental trophoblast in viruses infectious diseases. (C) 2016 Elsevier Inc. All rights reserved.