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Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

Fu M, Chen Y, Xiong X, Lan D, Li J - PLoS ONE (2014)

Bottom Line: The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing.The cells expressed more antimicrobial peptides upon S.aureus invasion.Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, China.

ABSTRACT
This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

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Real-time PCR analysis of antimicrobial peptides and apoptotic factors.CG: Control group without treatment; and S.aureus treated: cells were invaded by S.aureus. Each bar shows the mean ± SE of three independent experiments. GAPDH was selected as the internal control gene. The symbol “*” indicates significant variation (p<0.05) compared with the control cells, and “**” indicates highly significant variation (p<0.01).
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pone-0113669-g009: Real-time PCR analysis of antimicrobial peptides and apoptotic factors.CG: Control group without treatment; and S.aureus treated: cells were invaded by S.aureus. Each bar shows the mean ± SE of three independent experiments. GAPDH was selected as the internal control gene. The symbol “*” indicates significant variation (p<0.05) compared with the control cells, and “**” indicates highly significant variation (p<0.01).

Mentions: To investigate the reaction of YMECs to S. aureus invasion, we performed real-time PCR to detect the expression levels of antimicrobial peptides (i.e., TAP and BNBD5) and apoptotic factors (i.e., BAX and BCL-2). The expression of antimicrobial peptide gene was induced when cells were challenged with S. aureus (Figs. 9A and 9B). Moreover, the expression of the apoptotic protein BAX significantly increased (Fig. 9C), whereas that of the anti-apoptotic protein BCL-2 decreased compared with those of the control group (Fig. 9D). Flow cytometry assay (AnnexinV–FITC/PI) was performed to determine whether apoptosis occurs in S. aureus infection-induced YMECs (Fig. 10). For infected YMECs, early apoptotic cells (annexin V+PI−) and post-apoptotic necrosis (annexin V+PI+) significantly increased compared with those of the control group (p<0.05).


Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

Fu M, Chen Y, Xiong X, Lan D, Li J - PLoS ONE (2014)

Real-time PCR analysis of antimicrobial peptides and apoptotic factors.CG: Control group without treatment; and S.aureus treated: cells were invaded by S.aureus. Each bar shows the mean ± SE of three independent experiments. GAPDH was selected as the internal control gene. The symbol “*” indicates significant variation (p<0.05) compared with the control cells, and “**” indicates highly significant variation (p<0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257549&req=5

pone-0113669-g009: Real-time PCR analysis of antimicrobial peptides and apoptotic factors.CG: Control group without treatment; and S.aureus treated: cells were invaded by S.aureus. Each bar shows the mean ± SE of three independent experiments. GAPDH was selected as the internal control gene. The symbol “*” indicates significant variation (p<0.05) compared with the control cells, and “**” indicates highly significant variation (p<0.01).
Mentions: To investigate the reaction of YMECs to S. aureus invasion, we performed real-time PCR to detect the expression levels of antimicrobial peptides (i.e., TAP and BNBD5) and apoptotic factors (i.e., BAX and BCL-2). The expression of antimicrobial peptide gene was induced when cells were challenged with S. aureus (Figs. 9A and 9B). Moreover, the expression of the apoptotic protein BAX significantly increased (Fig. 9C), whereas that of the anti-apoptotic protein BCL-2 decreased compared with those of the control group (Fig. 9D). Flow cytometry assay (AnnexinV–FITC/PI) was performed to determine whether apoptosis occurs in S. aureus infection-induced YMECs (Fig. 10). For infected YMECs, early apoptotic cells (annexin V+PI−) and post-apoptotic necrosis (annexin V+PI+) significantly increased compared with those of the control group (p<0.05).

Bottom Line: The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing.The cells expressed more antimicrobial peptides upon S.aureus invasion.Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, China.

ABSTRACT
This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

Show MeSH
Related in: MedlinePlus