Effects Of Lipoteichoic Acid On Tight Junction Protein In Mammary Gland Epithelial

Mastitis to the dairy industry caused great economic losses,staphylococcus aureus is caused by dairy cow mastitis one of the main pathogens.Blood-milk barrier is a natural barrier against of mammary gland pathogenic microorganisms invasion,consisting mainly of tight junction structure.In this study,we examined the effect of lipoteichoic acid on the tight junction protein of mammary epithelial cells to provid the experimental basis for revealing the effect of Staphylococcus aureus on the blood-milk barrier of dairy cows.In vitro test,the bovine mammary epithelial cells were isolated and purified by tissue culture and gradient trypsin digestion method.Transwell cells were used to establish the mammary epithelial barrier model.The test were divided into 5 groups--A,B,C,D and E.Group A was the control group,B,C,D and E were LTA stimulation group.The stimulus quantity were0.01?g/m L,0.1?g/m L,1?g/m L,10?g/m L,respectively.The permeability of mammary epithelial b arrier model would be measured 24 hours later.The expression of Occludin,Claudin-1 and ZO-1m RNA was detected by real-time fluorescence quantitative PCR.Vivo tests were performed by injecting LTA into the nipple catheter.The amount of stimulation was consistent with that in vitro.The mouse mastitis model was established.Histopathological changes were observed by HE staining,the expression of TNF-? was detected by Elisa method,the quantitative protein expression of ZO-1 ? Occludin and Claudin-1 was detected by Western-blot,the location expression of Occludin and Claudin-1 was detected by immunofluorescence.The test results:(1)Immunofluorescence assay showed that the expression of keratin-18 was positive,indicating that the mammary gland epithelial cells were successfully obtained by tissue culture.(2)The mammary epithelial cells were cultured on Transwell chamber PET membrane,and the transmembrane resistance tended to be stable after 96 h,forming a monolayer cell HE staining and Elisa results showed that mouse mastitis model was successfully constructed by stimulating mouse mammary gland tissue with 1 ?g and 10 ?g LTA.(4)Permeability results showed a significant change in LTA permeability at 0.1?g/ m L(P < 0.05)compared with the control group,with significant changes in LTA permeability at 1 ?g / m L and 10 ?g / m L(P <0.01).(5)The q RT-PCR results showed that compared with the control group,the expression of Claudin-1 m RNA did not change,and the expression of Occludin and ZO-1 m RNA decreasedwith the increase of LTA concentration.(6)Western blot showed that the expression of Occludin and ZO-1 protein was consistent with the m RNA expression trend,compared with the control group,10 ?g LTA Claudin-1 protein decreased significantly(P < 0.05).(7)Immunofluorescence results showed that Occluidn and Claudin-1 were localized on the cell membrane,and 10 ?g LTA could cause Occludin fluorescence intensity treduction.In summary,LTA of Staphylococcus aureus can increase the permeability of mammary epithelial cells and reduce the expression of Occludin and ZO-1.