Biological characterization of bovine mammary epithelial cell lines immortalized by HPV16 E6/E7 and SV40T
Biological characterization of bovine mammary epithelial cell lines immortalized by HPV16 E6/E7 and SV40T
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HPV16 E6/E7 和 SV40T 永生化牛乳腺上皮细胞系的生物学特性
DOI：
10.1007/s11626-016-0063-8
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发表时间：
2016-10-01
影响因子：
2.1
通讯作者：
Zhao, Guo-Qi
中科院分区：
文献类型：
--
作者：
Zhan, Kang;Lin, Miao;Zhao, Guo-Qi
原代牛乳腺上皮细胞并不是理想的长期研究模型，因为原代细胞在体外增殖次数有限，并进入称为细胞复制衰老的生长停滞阶段，因此必须在体外建立永生化的牛乳腺上皮细胞（BMEC）。更重要的是，永生化细胞和凋亡细胞之间的关系的机制仍然是未知的BMEC。因此，我们试图阐明永生化细胞逃避凋亡信号通路的机制。这些细胞成功地永生化，没有任何衰老的迹象。BMEC和E6 E7永生化细胞在培养6 d后达到最大数量。在这一点上，有显着更多的E6 E7永生化细胞比原代BMEC（P< 0.01）。E6 E7和SV 40 T永生化细胞的群体倍增时间在48和72 h最低。我们未能检测到BMEC中上皮细胞标记物E-钙粘蛋白的表达;然而，永生化细胞的E-钙粘蛋白表达较低。β-catenin在永生化细胞中的表达明显高于BMEC（P< 0.01）。检测到胱天蛋白酶-3、胱天蛋白酶-9和聚ADP-核糖聚合酶（PARP）;然而，未观察到胱天蛋白酶-3和PARP的裂解。我们的数据表明，caspase-9，caspase-3和PARP的表达不足以使永生化细胞凋亡，并提示E-cadherin和β-catenin可能是癌症发展的重要指标。
Primary bovine mammary epithelial cells are not ideal models for long-term studies, because primary cells undergo a limited number of proliferations in vitro and enter into a growth-arrest stage called cell replicative senescence; we therefore must establish the immortalized bovine mammary epithelial cells (BMECs) in vitro. More importantly, the mechanisms of the relationship between immortalized and apoptotic cell remain unknown in BMECs. We therefore sought to elucidate the mechanisms of which immortalized cells escape the pathway of apoptotic signal. These cells were successfully immortalized without any signs of senescence. The maximum number of BMEC and E6E7 immortalized cells were reached after 6 d of culture. At this point, there were significantly more E6E7 immortalized cells than primary BMECs (P< 0.01). The population-doubling times of the E6E7 and SV40T immortalized cells were lowest at 48 and 72 h. We failed to detect the expression of the epithelial cell marker E-cadherin in BMECs; however, immortalized cells had low expression of E-cadherin. The expression of β-catenin was markedly expressed in immortalized cells than in BMECs (P< 0.01). Caspase-3, caspase-9, and poly ADP-ribose polymerase (PARP) were detected; however, the cleavage of caspase-3 and PARP was not observed. Our data demonstrate that the expressions of caspase-9, caspase-3, and PARP are not sufficient for the apoptosis of immortalized cells and suggest that E-cadherin and β-catenin might be an important indicator of the development of cancer.