Establishment of an immortalized stromal cell line derived from human Endometriotic lesion.
Establishment of an immortalized stromal cell line derived from human Endometriotic lesion.
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源自人子宫内膜异位病变的永生化基质细胞系的建立
DOI：
10.1186/s12958-020-00669-x
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发表时间：
2020-11-23
期刊：
影响因子：
--
通讯作者：
Chen QX
中科院分区：
文献类型：
--
作者：
Huang ZX;Wu RF;Mao XM;Huang SM;Liu TT;Chen QH;Chen QX
背景子宫内膜异位症是一种具有明显雌激素依赖性和炎症反应特征的妇科良性疾病。子宫内膜异位症原代间质细胞存在寿命短、激素脱分化和细胞因子反应性等问题，限制了其在子宫内膜异位症研究中的应用。本研究的目的是建立和鉴定永生化人子宫内膜异位基质细胞（ihESCs.MethodsThe子宫内膜异位标本来自卵巢子宫内膜异位症患者，原代子宫内膜异位基质细胞分离自子宫内膜异位组织。用慢病毒感染原代细胞，建立端粒酶逆转录酶（hTERT）诱导的永生化细胞。通过定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹法检测mRNA和蛋白质的定量。CCK-8法和EdU标记法用于评估ihESCs的生长。核型分析检测细胞染色体。结果经慢病毒感染后，hESCs持续高表达hTERT，并显著延长了hESCs的生存期，传代31代。ihESC的形态、增殖和核型保持不变。上皮间质转化（EMT）标志物，雌激素代谢蛋白和雌激素/孕激素受体（ER和PR）的表达没有改变。此外，雌激素的处理增加了ihESCs的增殖和EMT。脂多糖（LPS）和IL-1β可明显诱导炎症反应。ihESCs的克隆形成能力与原代细胞一致，远低于石川细胞。此外，裸鼠致瘤性试验表明，ihESCs是无法触发tumor formation.ConclusionThis研究建立和特点的永生化的肿瘤基质细胞系，表现出较长的寿命，并保持原代细胞的细胞形态和生理功能。永生化细胞对雌激素和炎症反应保持正常反馈。此外，永生化细胞不具有致瘤能力。因此，人胚胎干细胞可作为研究子宫内膜异位症发病机制的体外细胞工具。
BackgroundEndometriosis is a benign gynecological disease with obviously feature of estrogen-dependence and inflammatory response. The applications of primary endometriotic stromal cells in research of endometriosis are restricted for short life span, dedifferentiation of hormone and cytokine responsiveness. The objective of this study was to establish and characterize immortalized human endometriotic stromal cells (ihESCs).MethodsThe endometriotic samples were from a patient with ovarian endometriosis and the primary endometriotic stromal cells were isolated from the endometriotic tissues. The primary cells were infected by lentivirus to establish telomerase reverse transcriptase (hTERT)-induced immortalized cells. Quantification of mRNA and proteins was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot. CCK-8 assay and EdU labeling assay were assigned to assess the growth of ihESCs. Karyotype assay was performed to detect the chromosomes of ihESCs. Colony formation assay and nude mouse tumorigenicity assay were used to evaluate colony-formation and tumorigenesis abilities.ResultsihESCs continuously overexpressed hTERT via infection of lentivirus and significant extended the life span reaching 31 passages. The morphology, proliferation and karyotype of ihESCs remained unchanged. The expression of epithelial-mesenchymal transition (EMT) markers, estrogen-metabolizing proteins and estrogen/progesterone receptors (ERs and PRs) were unaltered. Furthermore, the treatment of estrogen increased the proliferation and EMT of ihESCs. Lipopolysaccharides (LPS) and IL-1β remarkably induced inflammatory response. The clonogenesis ability of ihESCs was consistent with primary cells, which were much lower than Ishikawa cells. In addition, nude mouse tumorigenicity assay demonstrated that ihESCs were unable to trigger tumor formation.ConclusionThis study established and characterized an immortalized endometriotic stromal cell line that exhibited longer life span and kept the cellular morphology and physiological function as the primary cells. The immortalized cells remained normal feedback to estrogen and inflammatory response. Moreover, the immortalized cells were not available with tumorigenic ability. Therefore, ihESCs would be serviceable as in vitro cell tool to investigate the pathogenesis of endometriosis.
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影响因子：
64.5
作者：
Han SJ;Jung SY;Wu SP;Hawkins SM;Park MJ;Kyo S;Qin J;Lydon JP;Tsai SY;Tsai MJ;DeMayo FJ;O'Malley BW
通讯作者：
O'Malley BW
影响因子：
13.3
作者：
Reis, Fernando M.;Petraglia, Felice;Taylor, Robert N.
通讯作者：
Taylor, Robert N.
影响因子：
2.4
作者：
Bozdag, Gurkan
通讯作者：
Bozdag, Gurkan
影响因子：
7.5
作者：
Mari-Alexandre, Josep;Garcia-Oms, Javier;Gilabert-Estelles, Juan
通讯作者：
Gilabert-Estelles, Juan
影响因子：
4
作者：
Bozdag, Gurkan;Alp, Alpaslan;Gurgan, Timur
通讯作者：
Gurgan, Timur