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

Abstract

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.

CLC Number:

S826.3

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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Establishment of Sheep Immortalized Epididymal Epithelial Cell Line and Analysis of Its Biological Characteristics

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