The Effect Of Borrelia Burgdorferi Virulence Factor BmpA On Expression Of Myeloid Differentiation Factor 88(MyD88)in Macrophages

Objectives:Macrophages differentiate from THP-1(human monocyte)cells by phorbol-12-myristate-13-acetate(PMA)was used as experimental model.We used same concentration recombinant Borrelia burgdorferi membrane protein(rBmpA)to stimulate macrophages and then,we collected cells at different time to examine the expression level of mRNA and protein of myeloid differentiation factor(MyD88)which is a key linker in the Toll-like receptor signaling pathway.We hope to understand the mechanism of proinflammatory chemokine crisis induced by Borrelia burgdorferi virulence factor.Methods:1.THP-1 cell culture:THP-1 cells were cultured by 10%fetal bovine serum 1640 medium at 37 0C in a 5%CO2 incubator and then,cells were enlarge cultured with 3-4 days.2.THP-1 cell differentiation:The number of THP-1 cells was adjusted to 5 × 105 cells/mL and then,600 ng/mL PMA was added into it.The cell suspension was putted into a 24-well cell culture plate with 500 ?L/well at 37 ?,5%CO2 incubator for 24 hours to induce cells differentiation.3.The macrophages differentiate from THP-1 cells were treated by LPS and rBmpA:After THP-1 cells were differentiated to macrophages,the supernatant was removed and then,LPS and rBmpA were added into well.The cells were incubated in a 5%CO2 incubator for 6 hours,12 hour,24 hours and 48 hours.Cells were collected at each time.4.Examinations of mRNA and protein expression:cells were lysed by cell lysis solution,and the expression levels were examined by real-time PCR and western blot.Results:1.THP-1 cells suspended in 10%FBS 1640 medium and grew well.2.THP-1 cells successfully differentiated into adherent macrophages under the induction of 600ng/ml-PMA.3.After macrophages were treated by LPS and rBmpA,which presented morphological change.4.Most of the relative expression of MyD88 mRNA in macrophages were induced by rBmpA at different times presented statistically significant(P<0.05,P<0.01),which compared with normal control group.5.Most of the expression of MyD88 protein in macrophages were induced by rBmpA at different time presented statistically significant(P<0.05,P<0.01),which compared with normal control group.Conclusions:In macrophages,the MyD88 is a critical role in TLRs signal pathway when the inflammation was induced by rBmpA.