Isolation And Culture Of Bovine Mammary Epithelial Cells(pbMEC) And The Protective Effect Of Sodium Selenite Against Inflammatory Injury Induced By LPS On The PbMEC

Bovine mastitis is one of the most harmful diseases in dairy production. With development of dairy industry and the rising attention to public health, people pay more attention to the harm of bovine mastitis. Early diagnosis of bovine mastitis is very significancant, especially subclinical mastitis. Method of Prevent and Control mastitis has some shortcomings.TO find safe and effective Method of Prevent and Control of mastitis,and to evaluate the effection of drugs therapy and control to mastitis,following researches were made.:1Isolation and culture of bovine mammary epithelial cells(pbMEC)pbMEC was isolated by explant culture and enzyme digestion;different formula of enzyme and different time of digestion was used to isolate pbMEC.The result showed:(1)pbMFC seperated from explant after3days and grown massively after5days.pbMEC firstly seperated from the explant after7-9days,and grown massively after10days.pbMEC was like paving stones after2times of purifications,and could form Island-like structure.Acinar-like structures also could be formed.Results showed that pbMEC was successfully isolated.(2)Use0.25%trypsin and0.25%collagenase II to digest tissue to4hours under37℃was the most effctive way of digesion,and1×105cells per issue could be obtained.2The protective effect of sodium selenite against inflammatory injury induced by LPS on the pbMECCell activition was detected after stimulated by10μg/ml、50μg/ml、100μg/m、150μg/ml、200μg/ml LPS after36h,and by200μg/ml LPS for12h、24h、36h, respectively,to analyse cell injury induced by LPS;Cell activition was detected after stimulated by1μM、2μM、4μM、8μM、10μM、20μM、40μM、80μM Sodium selenite for3days,to investigate injury made by Se.Leakage rate of LDH and content of MDA of3groups:Se+LPS group,LPS group and control group were detected after LPS induced.The result showed that:(1)Cell acvivity dropped to59.6%after36hours of200p.g/ml LPS induction,which was significantly differeny from Se groups and control gruops (P<0.05),but activity of induction of12h and24h was not significantly differeny from Se groups and control gruops (P>0.05).The result explained that only200μg/ml LPS induce pbMEC to36could the injury of activity be made.(2)Activity of cells induced by1μM,2μM Se for1day,2daysand3days show no significant difference between control group (P>0.05),but it dropped to87.8%by induction of4μM Se for3days,which was significantly different from control gruop (P<0.05).The difference revealed that concentration of Se under2μM was safe to pbMEC.(3) LDH leakage rate of cells stimulated by200ug/ml LPS for36h significantly rised (P<0.05),but it showed no significant difference between10μg/ml、50μg/ml100μg/ml150μg/ml LPS groups (P>0.05).It showed that only200ug/ml LPS with stimulation of36h injured cells.(4)MDA content of LPS gruop rised significantly compared with control group (P<0.05).while Se group showed no significant difference between control groou,but signifacantly differed from LPS group.which explained that Se protected pbMEC form LPS injury.3The regulation of sodium selenite of TLRs、IL-1β and IL-8gene on pbMEC induced by LPSTLR2、TLR4、IL-1β、IL-8mRNA expression were detected by RT-PCR,the results showed:(1)TLR2、TLR4、IL-1β、IL-8mRNA expression of LPS group significantly rised compared with control group (P<0.05);(2)TLR2、TLR4、IL-1β、IL-8mRNA expression of Se group showed no significant difference between control group (P>0.05).It meant that LPS up-reguated pbMEC inflammation factors,while Se inhibited this LPS regulatin.