Establishment And Physiological Function Identification Of Bovine Mammary Epithelial Cells

In this study we used explant culture method to separate bovine mammary epithelial cells (BMEC), and adopted many methods to purified cells. These cells were identified for their morphology and physiological function. Real time PCR technique was employed to examine the mRNA expression for IL-6, IL-8and TNF-α by stimulation of16th passage cell with LPS, meantime we investigated the effects on milk protein synthesis and secrete of16th passage with lactogenic hormone and lysine. By this way, we thought that our lab had successfully established BMEC line with the normal functions, these works had provided more advantages for further establishing immortalized cell lines.Experiment1:A large number of primary BMEC could be gained from tissue culture, then purified cells were obtained by twice digestion and adherence, scraping and single-cell clone. The proliferation capacity of epithelial cells declined after25passages. The morphology and milk protein genes expressing of BMEC in vitro were detected by fluorescence immunocytochemical method, karyotype analysis and beta-casein expression. The results showed that these cells had a typical characteristic of the epithelial cells and expression cytokeration18of marker protein, and these cells could express low beta-casein gene.Experiment2:We selected suitable concentration of LPS which stimulated bovine mammary epithelial cell. Using real time PCR technique we got the result of mRNA expression for IL-6, IL-8, TNF-a. The result showed that BMEC stimulated by LPS for24h increased the mRNA levels of the genes of IL-6and IL-8, TNF-a expressed at the top of LPS for4h. The cell line had the valuable application, the cell line could use as a model to study mastitis mechanism, and they had an important role in the cell innate immunity.Experiment3:Relative quantitative real-time PCR was employed to examine the mRNA expression for beta-casein, alpha-lactalbumin and lactoferrin with the stimulation of different combination of hormone. The result showed that the mRNA levels of the genes of these proteins all were more significantly higher than those in control group when prolactin, hydrocortisone and insulin were in the presence. The same combination above-mentioned, addition of lysine, the mRNA levels of the genes had increased trend, but not changed significantly. In order to confirm the results of relative quantitative real-time PCR, we used the method of ELISA to analyse the protein expression level of beta-casein and lactoferrin. The results showed that these protein level expression patterns were accorded with mRNA levels. It suggested that bovine mammary epithelial cells did not transform and had the normal physiological function.