| Mammary gland is one of the few mammals that can repeat growth,functional differentiation and degeneration in mammals.It is an exocrine gland derived from the sweat gland.The main function is that produces milk to feed young offspring.The quality of breast development determines the growth and development of young animals and important for milk production.Estrogen promotes the proliferation of mammary epithelial cells,the extension of mammary ducts,and the initiation and maintenance of lactation.Therefore,it is of great significance to study the mechanism of estrogen on proliferation and phenotype maintenance of mammary epithelial cells.As a newly discovered estrogen membrane receptor in recent years,G-protein coupled receptor 30(GPR30)has been activated to mediate rapid non genomic effects to regulate cell function.Our lab's studies have found that estrogen played an important role in the proliferation and epithelial mesenchymal transition(EMT)of goat mammary gland epithelial cells.Whether estrogen can participate in these processes through the GPR30 signaling pathway require further study.In this experiment,We used proper method for dissociation of goat mammary epithelial cells from Guanzhong dairy goat mammary gland tissue in primary cell culture.and then the expression and location of GPR30 in goat mammary epithelial cells were determined.Finally,GPR30 specific agonist G1 and inhibitor G15 were used to investigate the mechanism of estrogen /GPR30 signaling pathway on the proliferation and the maintenance of epithelial phenotype of goat mammary epithelial cells.The main contents and results are as follows:(1)A serum-free tissue culture method was used to isolate and purify goat mammary epithelial cells.These cultured cells expression of keratin 18,no expression of keratin 14;RT-PCR detection of lactation related specific genes showed that the cells expressed ?-casein mRNA continuously in the serum-free culture system.In conclusion,we determined the cells that isolated from mammary gland tissue of goats were goat mammary epithelial cells.By immunofluorescence,the estrogen receptor GPR30 was expressed in the mammary epithelial cells,and the main expression site was located on the cytoplasmic membrane.RT-PCR andWestern Blotting results showed that the goat mammary epithelial cells continuously expressed GPR30 in serum-free culture system.(2)The effects of estrogen /GPR30 signaling pathway on the proliferation of goat mammary gland epithelial cells were studied by methods of cell counting and BrdU incorporation.The results showed that estrogen could promote the proliferation of goat mammary epithelial cells in a dose-dependent manner.In the culture system containing estrogen or estrogen receptor GPR30 agonist G1,activation of the estrogen /GPR30 signaling pathway that promote the proliferation of mammary epithelial cells in goats.The estrogen/GPR30 signaling pathway was not activated in the culture system containing GPR30 inhibitor G15 or without estrogen,its proliferative activity were significantly suppressed.It indicates that estrogen can mediate the proliferation of goat mammary epithelial cells through GPR30 signaling pathway.(3)The effects of estrogen /GPR30 signaling pathway on the EMT in goat mammary epithelial cells were studied by immunofluorescence,q-RT PCR and Western Blotting.The results showed that in the culture system containing estrogen or estrogen receptor GPR30 agonist G1,the estrogen /GPR30 signaling pathway was activated to maintain the epithelioid characteristics of goat mammary epithelial cells and maintain the high expression of E-cadherin mRNA,and the low expression of N-cadherin,Vimentin and Snail2 mRNA and protein.All these led to inhibit EMT in goat mammary epithelial cells.In the culture system containing G15 or without estrogen,the estrogen /GPR30 signaling pathway was suppressed.The goat mammary epithelial cells lose the morphological characteristics of epithelioid cells,show the morphological characteristics of interstitial cell like,and the low expression of E-cadherin,high expression of N-cadherin,Vimentin and Snail2.These caused occurrence EMT in goat mammary epithelial cells.It indicates that estrogen /GPR30 signaling pathway mediates the maintenance of epithelial phenotype and inhibits EMT in goat mammary epithelial cells.(4)Western Blotting was used to detect the expression of ERK 1/2(extracellular regulated protein kinases 1/2)that was the key signal molecule in the downstream of estrogen /GPR30 signaling pathway.The results showed that estrogen could activate ERK 1/2rapidly.The phosphorylation level increased rapidly in 15 min and lasted for at least 4 h.Estrogen can rapidly phosphorylate ERK 1/2 through the GPR30 signaling pathway.It indicates that ERK 1/2 is involved in the process of estrogen /GPR30 signaling pathway on the proliferation and epithelial phenotype maintenance of goat mammary epithelial cells. |
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