Establishment of Immortalized Porcine Mammary Epithelial Cell Line
CAO Jia-Ming1,*, KAN Yao2,*, LI Zhi-Yuan1, ZHONG You-Gang2,**, LI Qiu-Yan1,**
1 College of Biological Sciences, China Agricultural University, Beijing 100193, China; 2 College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
Abstract：Porcine mammary epithelial cells (PMECs) are highly differentiated in vitro and can maintain their unique morphological characteristics and function of producing and secreting milk proteins. Therefore, it is of great significance to establish a stable immortable PMECs line that can maintain the characteristics of PMECs for the study of the development, differentiation and lactation mechanism of porcine mammary gland. In this study, tissue block culture method and enzymatic digestion method were used to isolate and purify mammary epithelial cells from sows (Sus scrofa) at the end of pregnancy, and the cell culture method was optimized. Human telomerase reverse transcriptase (hTERT) gene was transfected into primary cultured pig mammary epithelial cells, by PCR detection, the immortalized hTERT-PMECs was obtained. The morphological observation of hTERT-PMECs cultured continuously for 60 generations showed that the type of hTERT-PMECs cells did not change after immortalization. The results of immunofluorescence staining showed that hTERT-PMECs could express keratin. After the induction of prolactin, hydrocortisone and Insulin-Transferrin-Selenium (ITS), hTERT-PMECs was identified by reverse transcription-PCR (RT-PCR), the result showed that hTERT-PMECs still had the function of secreting lactoprotein. In summary, this study successfully obtained the immortalized porcine mammary epithelial cell line hTERT-PMECs, and the immortalized cells maintained the same characteristics as the primary cells. This study provides basic data for the study of mammary gland development and lactation.
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