| TLR(Toll-like receptor) and NOD(Nucleotide-binding oligo merization domain protein)are pattern recognition receptors exist on cytomembrane or intracellular, which can recognize pathogenic microorganism and activate the signal pathway which would release inflammatory factor and involve in innate immune response. Fibroblasts are often inactive,which can support mammary gland tissue and repair injury in mammary gland. In recent years, fibroblasts were found to be activated by some stimulation, and the activated fibroblasts keep releasing inflammatory factors which turns acute into chronic. It is of great importance to do researches on the molecular mechanism of fibroblasts immunity. In this research, by detecting the expression of TLR and NOD in bovine mammary fibroblasts and studying the TLR and NOD pathways in cells, the innate immune of mammary fibroblasts was preliminary revealed which provides scientific basis for further research on bovine mammary fibroblasts immune mechanism.1. Culture and Isolation of bovine mammary fibroblastsFibroblasts were tissue cultivated and continuously purified with cell-suspension culture by the different tryspin sensitivity of fibroblasts and epithelials cells. The shapes and growth of cells were observed every day, as well as the drawing of growth curve. The result showed that fibroblasts began growing outside tissues since 3-5d, and epithelials cells outside growing could be observed in the 7th day. There were obvious boundary of the two cells which lead a clearly growth of each.3-5 times purification with cell-suspension culture, purified fibroblasts were got. It was observed by Giemsa staining that the cells were long spindle, smooth edge, loosely arranged, which could clearly distinguished from epithelials cells.2. mRNA Expression and semi-quantitative analysis of bovine mammary fibroblastsExpression of TLR1-TLR10 mRNA in spleen was analyzed by RT-PCR. Expression of TLR1-TLR10 mRNA in mammary fibroblasts was analyzed too. It was showed that TLR1-TLR10 all expressed in spleen and fibroblasts, among which expression of TLR1, TLR3, TLR4 and TLR5 were quite high and that of TLR2, TLR6, TLR7, TLR8, TLR9 and TLR10 were relatively low in mammary fibroblasts(P<0.05). The results demonstrated that TLR1～TLR10 had differential expression in bovine mammary fibroblasts.3. NOD mRNA expression and semi-quantitative analysis of mammary fibroblastsExpression of NOD1 and NOD2 mRNA in spleen was analyzed by RT-PCR.With semi-quantitative RT-PCR, expression of NOD1 and NOD2 gene mRNA in mammary fibroblasts were analyzed. Strips of NOD 1 and NOD2 were amplified in electrophoresis and expression value of NOD2 was higher than that of NOD1(P<0.05).It is inditcated that NOD1 and NOD2 had differential expression in bovine mammary fibroblasts.4. Expression detection of relevant genes of TLR and NOD pathway and its downstream inflammatory factors in mammary fibroblastsWith RT-PCR, expression of mRNA of MyD88, TRIF, TRAF6, IRAK1, IRAK4 and NF-κB(p65) were firstly detected, then detection of mRNA expression of IL-6, IL-8 and TNF-α were also done. Result showed that, adaptor protein MyD88 TRIF, TRAF6, IRAKI and IRAK4 of TLR signaling pathway, transcription factor NF-κB(p65) of TLR and NOD signaling pathway and inflammatory factor IL-6, IL-8 and TNF-α were all expressed in mammary fibroblasts. The result demonstrated that TLR and NOD signaling pathway may exsist in bovine mammary fibroblasts.5. Effect of PAMP induction on expression of TLR4, NOD2, IL-6, IL-8 and TNF-α gene mRNA in bovine mammary fibroblastsMammary fibroblasts were induced respectively by lipid A(TLR4 ligand) and MDP(muramyl dipeptide)(NOD2 ligand), and expression value change of mRNA of TLR4(or NOD2), IL-6, IL-8 and TNF-α were detected by RT-PCR. Result showed that mRNA level of TLR4, IL-6, IL-8 and TNF-αmRNA in lipid A group were significantly higher than that of control group(P<0.05); mRNA level of NOD2, IL-6, IL-8 and TNF-α in MDP group was much higher than that of control group(P<0.05). It was observed that expression of TLR4 and NOD2 and inflammatory factors would increase under the stimulation of lipid A and MDP, which indicated the stimulation of fibroblasts by lipid A and MDP might result in the release of inflammatory factors through TLR4 and NOD2 signaling pathway,and cause inflammatory. |
