| Objective①To investigate the effects of increasing stimuIating time on expression of TLR2 and TLR4 inperipheral blood mononuclear ceHs(PBMC) induced by Legionella in vitro.②To discuss related cytokines’（IL-6、TNF-α）role in infection of Legionella pneumophila.③To explore the ralationship between TLR4 (2244G'A) polymorphism and IL-6、TNF-α..Methods①Take 40mL of peripheral blood from healthy people (n = 30) by the Red Cross Blood Centerin Taiyuan City .before the separation of PBMC ,retain 5mL peripheral blood by density gradientcentrifugation in PBMC prepared of TLR4 the SNP genotyping, trypan blue staining to count thenumber of viable cells2×106cells per well seeded in 24-well plates at 37°C, 5% CO2 for 24 h.②For 24h, 24-well plates to remove the PBMC of healthy people is divided into normal controlgroup and Legionella stimulation, Legionella stimulation group were added to 500μL2×106/mL(MOI1 group), 2×107/mL (MOI10 group ) two different concentrations of Legionellasuspension, the three groups were to do multiple holes. 37℃, 5% CO2, incubation 2h, whenLegionella into the adherent cells, the supernatant was removed, with 1mL phosphate-bufferedsaline (PBS) after washing, add 1mL containing 10% FBS RPMI-1640 culture culture liquid,37°C, 5% CO2, respectively, 18、24、72 h, cells were harvested to extract total RNA. Eachgroup culture supernatant was stored at -80°C for IL-6, TNF-alpha detection.③Determinated TLR2, TLR4 and MyD88 mRNA expression levels using the Real-time RTPCR . the secreted content of cytokine IL-6、TNF-αin supernatant were analyzed byenzyme-linked immunosorbent assay(ELISA).④SNaPshot was used to detect the TLR4 SNP genotyping.⑤SPSS13.0 statistical software was used for data entry and analysis.Results①Factorial analysis showed that Leading effect of time have statistical significance (F=26.06，p<0.05) and there was interaction between time and concentration（F=11.39，p<0.05）。In 18hours, there was no difference between stimulated group and control groups. In 24 hours,The expressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml'2×107/ml legionella ,whichwere obviously higher than TLR2 mRNA expression in control group. In 72 hours, Theexpressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml'2×107/ml legionella ,whichwere obviously lower than TLR2 mRNA expression in control group.when the PBMCs arestimulated at the level of 2×106/ml'2×107/ml legionella.There is no statistical significance inexpression difference between expression stimulate group and control group of TLR4 mRNA（F=0.074，p=0.929）, the difference of each timing also shows statistical significance(F=12.782，p=0.000), and the interaction difference of grouping and timing shows no statisticalsignificance（F=0.948，p=0.440）.②The difference of each timing shows statistical significance(F=8.68，p<0.05), The differenceof IL-6 concentrations expression between MOI10 group and control group of Lung Tissue ofmice shows statistical significance（F=94.47，p<0.05）,and the interaction difference of groupingand timing shows no statistical significanc（eF=2.55，p>0.05）. The difference of each timing alsoshows statistical significance(F=3.324，p<0.05);The difference of TNF-αconcentrationsexpression between MOI10 group and control group shows statistical significance（F=4.511，p<0.05）, and the interaction difference of grouping and timing shows statistical significance（F=0.283，p>0.05）.③The concentration of TNF-αof GA/AA genetype group was higher than GG genetype groupsin healthy people, there was no significant difference（Z=-1.200, p =0.235）;. The concentrationof IL-6 of GA/AA genetype group was higher than GG genetype groups in healthy people, therewas no significant difference（Z=-1.609, p =0.116）.Conclusion①The study found that TLR2 may participate in resistance to Legionella infection, TLR4,MyD88 have not yet found participation of Legionella infections.②IL-6 and TNF-αwere related with Legionella infection.③GA/AA genotypes may be the protection factor with Legionella infection. |
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