Effects Of In-msflammation-related Fibroblasts Derived From Mastitic Bovine Mammary Tissueon Biological Characteristics And β-casein Secretion Of Mammary Epithelial Cells

Bovine mastitis is one of the most prevalent diseases, which is characterized by a mammary gland inflammatory response caused by metabolic and physiologic changes, trauma or, more frequently, contagious or environmental pathogenic microorganisms. Epithelial cells and fibroblasts, major compositions of the mammary tissue, play an important role in the development, lactation and involution of mammary gland and mammary-related diseases. It has been confirmed epithelial cells, macrophages, neutrophils, lymphocytes and other breast cells constitute the breast tissue defense system, which participate in cellular immune responses in the process of dairy cow mastitis. But the functions of normal fibroblast in the process of bovine mastitis have not been studied. The effects of fibroblasts derived from mastitis bovine mammary tissue on biological characteristics and lactation function of normal epithelial cells were investigated to clarify the role of fibroblasts in the process of bovine mastitis, and provide new ideas and directions for the prevention and treatment of bovine mastitis. The main work and results are as follows:Epithelial cells were isolated from mammary tissue of Holstein cows introduced to the slaughterhouse due to problems of fractured legs during the lactation, and the epithelial cells were respectively co-cultured with stromal fibroblasts derived from mastitis(Inflammation-related MSF,In-MSF) or normal mammary tissue(Normal MSF, N-MSF) after purification and identification. The proliferation test of CCK-8, cell scratch assay, and Annexin V-FITC apoptosis detection kit were used to investigate the effects of stromal fibroblasts from mastitis mammary tissue on the proliferation, migration and apoptosis of epithelial cells. RT-PCR was used to detect the expression of TNF –α, IL-6, and IL-8. Beat-casein secretion was measured by ELISA kit. The findings of this study could verify whether if fibroblasts are involved in the inflammatory response when the mammary gland is infected.The results showed In-MSF inhibited the cell proliferation and migration of normal mammary epithelial cells, did not induce apoptosis when compared to N-MSF. In-MSF could up-regulate the expression of TNF-α and IL- 8 in epithelial cells. No difference was observed in the IL-6 expression in epithelial cells co-cultured with In-MSF and N-MSF. Furthermore, In-MSF was able to inhibit β-casein secretion of epithelial cell compared to N-MSF.It can be concluded that fibroblasts participate in the process of the development of bovine mastitis, and they had an impacts on the biological characteristics and functions of epithelial cells. The findings of this study would help to clarify mastitis regulatory mechanism of immune and inflammatory response, and provide a new research direction for prevention and treatment of bovine mastitis.