永生化绵羊附睾上皮细胞系的建立及其生物学特性分析

doi:10.11843/j.issn.0366-6964.2019.09.009

摘要/Abstract

摘要： 旨在建立绵羊附睾上皮永生化细胞系，为进一步研究绵羊附睾功能调节机制提供基础。本研究采用酶消化法分离培养原代绵羊附睾上皮细胞，利用脂质体将pCI-neo-hTERT质粒转入绵羊附睾上皮细胞（SEECs），经G418筛选得到了细胞系hTERT-SEECs，免疫荧光法鉴定其角蛋白18（CK18）和人端粒酶逆转录亚基（hTERT）表达情况；RT-PCR检测其hTERT mRNA的表达；采用CCK-8法绘制细胞生长曲线检测其增殖能力；利用流式细胞仪检测其周期和细胞凋亡情况；核型分析检测其倍体情况；从mRNA和蛋白水平检测其谷胱甘肽过氧化物酶5（GPX5）和雄激素受体（AR）的表达情况。结果显示，原代绵羊附睾上皮细胞呈典型的铺路石状形态。hTERT成功转入绵羊附睾上皮细胞，传45代后，hTERT-SEECs仍呈铺路石状；hTERT-SEECs仍可稳定表达CK18和hTERT，并且拥有正常的二倍体核型；hTERT-SEECs增殖能力高于原代细胞，hTERT-SEECs处于G1期的细胞比例显著低于SEECs（P<0.05），处于S期细胞比例显著高于SEECs（P<0.05），且其活细胞率显著高于SEECs（P<0.05），凋亡率显著低于SEECs（P<0.05）；hTERT-SEECs和SEECs的GPX5和AR蛋白表达量差异不显著（P>0.05）。本研究所建立的hTERT-SEECs经长期培养后仍具有正常的附睾上皮细胞形态，增殖能力强并保留了附睾上皮细胞的生物学特性。

Abstract: This study aimed to establish sheep immortalized epididymal cell line and provide a basis for further studying the regulation mechanism of epididymis function in sheep. The primary sheep epididymal epithelial cells were isolated and cultured by enzymatic digestion. The pCI-neo-hTERT plasmid was transferred into the sheep epididymal epithelial cells(SEECs) by using liposome and the hTERT-SEECs was screened by G418. The cell expression of keratin 18 (CK18) and human telomerase reverse transcriptase(hTERT) were identified by immunofluorescence. The expression of hTERT mRNA was detected by RT-PCR.The cell growth curve was detected by CCK-8. The growth cycle and apoptosis status were detected by flow cytometry. The ploidy was detected by karyotype analysis. The mRNA and protein expression of glutathione peroxidase 5 (GPX5) and androgen receptor (AR) were detected. The results showed that the primary sheep epididymal epithelial cell exhibited native cobblestone morphology. hTERT was transferred successfully into the SEECs. After 45 passage, the hTERT-SEECs still showed cobblestone morphology, the CK18 and hTERT could be steadily expressed in hTERT-SEECs,and the cells were in normal diploid, the proliferation ability of hTERT-SEECs was higher than that of primary SEECs, the percentage of hTERT-SEECs in G1 phase were significantly less than that of SEECs (P<0.05), while the percentage of hTERT-SEECs in S phase was significantly higher than that of SEECs (P<0.05), the percentage of living hTERT-SEECs was significantly higher than that of SEECs (P<0.05), the apoptosis rate of hTERT-SEECs was significantly less than that of SEECs (P<0.05). There were no significant difference in GPX5 and AR protein concentrations between hTERT-SEECs and SEECs (P>0.05). After long term culture, the hTERT-SEECs established in this study can maintain normal epididymal epithelial cell morphology, have strong proliferative capacity and retain the biological characteristics of epididymal epithelial cells.

中图分类号:

S826.3

宋慧子, 栾兆进, 王兆琛, 杜炜, 赵勇超, 张家新. 永生化绵羊附睾上皮细胞系的建立及其生物学特性分析[J]. 畜牧兽医学报, 2019, 50(9): 1822-1831.

SONG Huizi, LUAN Zhaojin, WANG Zhaochen, DU Wei, ZHAO Yongchao, ZHANG Jiaxin. Establishment of Sheep Immortalized Epididymal Epithelial Cell Line and Analysis of Its Biological Characteristics[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1822-1831.

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