Establishment of an immortalized human extravillous trophoblast cell line by retroviral infection of E6/E7/hTERT and its transcriptional profile during hypoxia and reoxygenation
Establishment of an immortalized human extravillous trophoblast cell line by retroviral infection of E6/E7/hTERT and its transcriptional profile during hypoxia and reoxygenation
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DOI：
10.3892/ijmm_00000121
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发表时间：
2009-02-01
影响因子：
5.4
通讯作者：
Tanaka, Tadao
中科院分区：
文献类型：
--
作者：
Omi, Hiroko;Okamoto, Aikou;Tanaka, Tadao
由于缺乏合适的细胞模型，对人滋养层细胞功能的研究受到很大限制。我们的目的是建立正常人滋养层细胞系，具有较长的寿命，并提供一个理想的体外细胞模型。原代人滋养层细胞来源于在妊娠第7周进行选择性流产的胎盘。通过用含有16型人乳头瘤病毒E6和E7与人端粒酶逆转录酶（hTERT）组合的逆转录病毒表达载体感染使细胞永生化。通过使用一组抗体的免疫细胞化学、Western印迹、实时RT-PCR、侵袭测定、明胶酶谱、核型分析和裸鼠测定来进行细胞系的表征。采用基因芯片技术分析了缺氧（1%O-2，1 h）和复氧（20%O-2，6 h）条件下的基因表达谱。免疫细胞化学显示绒毛外滋养层细胞表型的hCG β，细胞角蛋白7，HLA-G和CD 9阳性染色。明胶酶谱法检测MMP-2和MMP-9的分泌。核型分析显示，该细胞的染色体数目在46 - 48之间，几乎正常，裸鼠致瘤性为110。使用寡核苷酸阵列检测到43个基因在缺氧过程中可逆的上调或下调。这种新的永生化细胞系HChEpC 1b是研究绒毛外滋养层功能的有用模型。
Investigation into the function of human trophoblasts has been largely restricted by a lack of suitable cell models. We aimed to produce normal human trophoblast cell lines with a long lifespan and to provide an ideal in vitro cell model. Primary human trophoblast cells were derived from a placenta that had undergone elective abortion at the 7th week of C,gestation. The cells were immortalized by infection with retroviral expression vectors containing the type 16 human papillomaviruses E6 and E7 in combination with human telomerase reverse transcriptase (hTERT). Characterization of the cell line was performed by immunocytochemistry using a panel of antibodies, Western blotting, real-time RT-PCR, an invasion assay, gelatin zymography, karyotype analysis and a nude mouse assay. Gene expression profiles under hypoxia (1% O-2, 1 h) and subsequent reoxygenation (20% O-2, 6 h) were analyzed using cDNA microarray. Immunocytochemistry revealed an extravillous trophoblastic phenotype by positive staining for hCG beta, cytokeratin 7, HLA-G and CD9. A transwell insert invasion assay showed the invasiveness of this cell line and gelatin zymography detected the secretion of MMP-2 and MMP-9. Karyotype analysis exhibited an almost normal chromosomal number which ranged from 46 to 48 and the cells showed 110 tumorigenecity in a nude mouse assay. Forty-three genes showing reversible up- or down-regulation during hypoxia were detected using an oligonucleotide array. This newly immortalized cell line, HChEpC1b, is a useful model for the Study of extravillous trophoblast function.