| Firstly, to reveal the internal cause of high incidence of mastitis during periparturient period, mRNA expression of inflammation related genes in mammary tissues and antioxidant indicators in plasma were determined at different reproductive stages of sows. Then, the in vitro model of mammary epithelial cell was established and characterized. At last, LPS was used to challenge mammary epithelial cells. Inflammation related genes were determined in mammary epithelial cell after LPS challenge. Thus, the establishment of mammary inflammation model in vitro was explored.1 Expression of inflammation related genes in mammary tissues and related indicators in blood of sows at different reproductive stages.Sampling of mammary tissues was conducted at day 90 of gestation, parturition and day 28 and 35 after parturition with Biopsies instrument and a Core Tissue Biopsy Needle. Mammary samples were then immediately frozen in liquid nitrogen and stored at-20℃ until analysis. Mammary mRNA expression of IL-1β at parturition was significantly (P< 0.05) lower than that at day 90 of gestation. And from parturition to day 35 after parturition, the mammary mRNA expression of IL-1β tended to go on an increase. At parturition, the mammary mRNA expression of IL-8 was 54.7% lower than that at day 90 of gestation. And the mammary mRNA expression of IL-8 at parturition was significantly (P< 0.05) lower than that at day 35 after parturition. From parturition to day 35 after parturition, the mammary mRNA expression of IL-8 also tended to go on an increase. The mammary mRNA abundance of TNF-a at parturition was significantly (P< 0.05) lower than that at day 90 of gestation and day 28 and 35 after parturition. Notably, the mammary mRNA abundance of TNF-a at day 35 after parturition was significantly (P< 0.05) higher than that at day 28 after parturition. Moreover, the mammary mRNA abundance of LBP at day 90 of gestation was significantly (P< 0.05) lower than that at parturition and day 28 and 35 after parturition. Consequently, the mammary mRNA abundance of IL-1β, IL-8 and TNF-α was lowest at parturition compared with that at the other reproductive stage and would increase after parturition. The mammary mRNA abundance of LBP was lower before parturition and would increase after parturition. Reproductive stages have no effect on plasma concentration of FFA and a-LA. At parturition, sows had a significant (P< 0.05) lower plasma concentration of a-tocopherol than at day 90 of gestation and day 28 after parturition. Sow plasma concentration of MDA at parturition tended (P< 0.1) to be greater than at day 90 of gestation, and that at day 28 after parturition significantly (P< 0.05) higher than at day 90 of gestation while had no difference with parturition..2 Primary culture and characterization of porcine mammary epithelial cell.Mammary tissues were collected after slaughter and subjected to tissue block culture. The growth media included 10% FBS, insulin, EGF and hydrocortisone. The cells present the typical morphological characters of epithelial cells by observation under an inverted microscope. The cells were then characterized by immunohistochemistry and they were positive to pan keratin reflecting that we obtained porcine mammary epithelial cells. And the growth curves indicated that cells grew in good condition.3 The effect of LPS on mRNA expression of inflammation related genes in porcine mammary epithelial cell.After grown to 80% confluence, porcine mammary epithelial cells were digested, counted and then seeded in two six-well plates. Total RNA was harvested from cells after incubation with LPS or not for 24 hours. QPCR were used to determine mRNA expression of inflammation related genes. Pictures of cells reflected no differences between control group and LPS group. However, LPS treated cells had very significantly greater (P< 0.01) mRNA expression of IL-8 and significantly higher mRNA expression of IL-1β than control cells. IL-8 and IL-1β mRNA expression in LPS group was 2.2 fold and 3 fold of that in control group. LPS had the trend (P< 0.1) to increase mRNA expression of PPAR-γ. LPS had no effect on TLR4 mRNA abundance... |
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