Studies On The Effect Of TGF-β1,bFGF And PDGF In Bovine Mammary Fibrosis

Bovine mammary fibrosis were a common pathological process mainly caused by mastitis. As a major diseases of dairy cow, the incidence and infection rates of bovine mastitis were higher both at home and abroad, could leading to various degree of mammary fibrosis. During the process of mammary gland fibrosis, the milk yield were decreased, in seriously may resulting in the breast hardening, then lossing the ability of lactating milk, lead to serious losses of dairy industry.The tissues and organs fibrosis were mainly depends on the deposition, decomposition of the extracellular matrix (ECM) and apoptosis of the related cells, which were closely related to cytokines such as transforming growth factor-P (TGF-β), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF). Currently, the research were mainly focus on the liver and kidney fibrosis, while rarely about the mammary fibrosis. In addition to the stromal fibroblasts producing ECM in fibrosis, whether the mammary epithelial cells could or not transdifferetiation into myofibroblasts then produce ECM remains unclear. It is reported that the a-smooth muscle actin (a-SMA), connective tissue growth factor (CTGF) and Collagen I (COL I) were the three main indicators during the epithelial-mesenchymal transition (EMT). Therefore, detected the changes of the three indicators could determine the role of the cytokines in the process of fibrosis.To obtain stability purified bovine mammary epithelial cells and fibroblasts in vitro, the mammary tissue sample were collected from Holstein cows during late gestation or lactation by surgery. After isolated the mammary alveoli, the mammary cells were cultured by tissue explants method in vitro. Then bovine mammary epithelial cells and fibroblasts were purified and cultured separately by different time of trypsin-EDTA digestion and adherent method, and the purity of the cells were identified by immunocytochemistry method. To explore the effect of the_three_cytokine_of TGF-β1, bFGF and PDGF on the bovine mammary epithelial cells and fibroblasts, the two kind of cells were incubated with various concentrations of exogenous TGF-β1(0,1,5,10ng/ml), bFGF(0,5,10,20ng/ml) and PDGF-BB(0,5,10,20ng/ml) separately,12h,24h,48h,72h later, the total RNA and protein were extracted from the cells, then the changes of the mRNA relative expression of the three transdifferentiation indicators (CTGF, α-SMA and COLIα1) were determined by Real-Time quantitative RT-PCR with P-actin as an internal reference gene, the PCR product were analysis by sequencing., and the corresponding protein were determined by Western blot method.The results showed that purified bovine mammary epithelial cells were mainly in polygonal, with round or oval nucleus, the nucleolus were visible and clear, which mainly in cobblestone or paving stone-like growth, and also could secret milk drop, with cytokeratin-18reaction positive, vimentin negative, while purified bovine mammary fibroblasts were mainly in spindle, in spiral or radial-shaped growth, with cytokeratin-18reaction negative, vimentin positive. After purification, the purity of both cells could exceed ninety-five percent, which could meet the requirement of following experiments. Exogenous TGF-β1could effetive increase the expression of a-SMA, CTGF, COLlα1mRNA and a-SMA protein both in the bovine mammary epithelial cells and fibroblasts, in a certain dose-effect way. Exogenous bFGF could increase the expression of CTGF mRNA both in the bovine mammary epithelial cells and fibroblasts, and mainly increase the expression of COL I al mRNA in the bovine mammary fibroblasts, while have no effect on the expression of COL I al mRNA in the bovine mammary epithelial cells, higher concentrations of bFGF have a inhibited tendency on the expression of a-SMA mRNA both in the bovine mammary epithelial cells and fibroblasts, which were inconsistent with the result of the protein expression. PDGF-BB mainly play a positive role on the expression of CTGF and COL I al both in the bovine mammary epithelial cells and fibroblasts, and could promote the expression of a-SMA protein in the bovine mammary fibroblasts, the expression of a-SMA mRNA were increased with lower concentration of PDGF-BB in the bovine mammary fibroblasts, while have a inhibited tendency with higher concentration of PDGF-BB on the expression of a-SMA mRNA in the bovine mammary epithelial cells, which were inconsistent with the results of the protein expression. The aim gene product of CTGF, a-SMA and COL I al amplified from the bovine mammary epithelial cells and fibroblasts were confirmed to be CTGF, a-SMA and COL I al gene from bovine by sequencing.The study confirmed that the two stably growth cell lines of bovine mammary epithelial cells and fibroblasts could be cultured and purified in vitro by the block tissue explants method combined with the different time of trypsin-EDTA digestion and the adherent method. TGF-β1could promote the expression of a-SMA, CTGF mRNA and COLlal mRNA and a-SMA protein both in the bovine mammary epithelial cells and fibroblasts, indicated that TGF-β1could promote transdifferentiation of the bovine mammary epithelial cell and generation of the ECM. Exogenous bFGF could increase the expression of CTGF mRNA both in the bovine mammary epithelial cells and fibroblasts, and mainly promote the expression of COL I α1mRNA in the bovine mammary fibroblasts, while have no effect on the bovine mammary epithelial cells, higher concentrations of bFGF have an inhibited tendency on the expression of a-SMA mRNA both in the bovine mammary epithelial cells and fibroblasts. Exogenous PDGF-BB mainly promote the expression of CTGF and COL I al mRNA both in the bovine mammary epithelial cells and fibroblasts, and promote the expression of a-SMA protein in the bovine mammary fibroblasts, the expression of a-SMA mRNA were increased with lower concentration of PDGF-BB in the bovine mammary fibroblasts, while were inhibited with higher concentration of PDGF-BB in the bovine mammary epithelial cells. Thus all the three cytokines have some certain effect on the expression of the transdifferentiation indicators both in the bovine mammary epithelial cells and fibroblasts, laid a foundation for the further study of the mechanisms and the treatment of bovine mammary fibrosis.