Human Keratinocytes Infected With Staphylococcus Aureus,the Changes Of Toll-iike Receptor 2 And Beta-defensin-3 In Cells

Objective:At present,the research on staphylococcus aureus(s.aureus)infection of human keratinocytes mainly focuses on the inhibition and killing effect of antimicrobial peptides on s.aureus,but there are few studies on the toll-like receptor and beta-defensin produced by the infected keratinocytes.In this experiment,by establishing an in vitro model of human keratinocytes(HaCaT cells)infected with s.aureus,the effect of s.aureus on the vitality of HaCaT cells was observed,and the expression changes of toll-like receptor 2(TLR2)and beta-defensin-3(hBD-3)in the infected cells were detected,and the immune mechanism of TLR2 and hBD-3 was preliminarily discussed to reveal the pathogenesis of Staphylococcus aureus after skin infection.Method:HaCaT cells inoculated in medium and wait until 80%cell aggregation,0.25%trypsinization,the supernatant was discarded and cell were resuspended in medium,then took count of the number of cells,adjust the cell concentration to 0.5×10~4/ml.S.aureus inoculated in the plate medium for 24h.Photographic record and observe its growths.A single colony was inoculated in liquid culture medium.After 16~18h,the bacteria were collected and resuspended in a DMEM base medium,the absorbance value of the bacterial liquid was measured at the wavelength of 660nm,and the concentration of the bacteria was calculated(0.8004×10~9 CFU/ml).Set up the blank control group and s.aureus treatment group.The MOI(multiplicity of infection=bacterial count?cell volume)of s.aureus treatment groupwasdividedinto1?20,1?10,1?1,3?1,10?1,20?1,50?1,100?1,and200?1.The HaCaT cells were inoculated at 96 orifice plate,each group of 5 compound pores,cultured infected cells for 8h,added CCK8 solution,cultured 2h in constant temperature incubator,and measured the absorbance of each group at the wavelength of 450nm of the Enzyme-linked immune detector,and the cell vitality was detected.MOI=20?1 group was more than half of cell death,its MOI was choosed as the optimum strain concentration.Repeated to set up the blank control group and MOI=20?1 group.The cells were inoculated with 6 orifice plates,and each group of 3 polypores was cultured for 8h to collect total RNA.The expression levels of intracellular TLR2 and hBD-3 mRNA were detected by RT-PCR..Design primers,PCR reaction conditions were set as:95?predenaturation 10min,95?denatured 30s,44?annealed 30s,72?extension 1min,34cycles,72?extension 10min.The PCR product was performed by 1.2%agarose gel electrophoresis.The electrophoretic images of each group were scanned to the computer to determine the integrated optical density of each strip.Results:1.The Result of CCK8:within 8 hours,as the concentration of bacteria increased,cell vitality gradually decreased,and the concentration of HaCaT cells in the MOI=20?1 group was more than half of the death.2.The Result of the Integrated Optical Density:compared with the blank control group,in the infected cells,the TLR2 mRNA increased by 64.369%and the hBD-3 mRNA increased by 160.367%.The expression of the two was positively correlated,and the expression of hBD-3 was increased by 95.998%more than that of TLR2(P<0.05).Conclusion:1.The infection of s.aureus can cause a decrease in the vitality of keratinocytes.2.The expression of intracellular TLR2 mRNA and hBD-3 mRNA is increased in human keratinocytes..The increase of TLR2 can further stimulate the higher expression of hBD-3.Review:Wesimplyanalysistherelationshipbetween s.aureus,keratinocytes,TLR,TLR2,?-defensin and hBD-3.And the immunological mechanism of TLR2 and hBD-3 in the keratinocytes infected by the staphylococcus aureus was preliminarily discussed.