| Objective①To explore the conditions of establishment of mice Legionella infection model.②To initially stated TLR2, TLR4's action in Legionella infection mechanism, look into the molecular targets of effects against Legionella infection and provide evidence for Legionella prevention and control.③To discuss related cytokines'(IL-6,TNF-α)role in mice infection of Legionella pneumophila.Methods1. Bought 80 SPF level 6-7 week BALB / C mice from Beijing Vital River Laboratory Animal Technology Company, N=8, male/female.40 mice intranasally inoculated with Legionella 50ul 1.2×109/ml bacterial suspension were as infection group, to establish Legionella infection model. The concentration of Legionella bacteria suspension was determined by Maxwell turbidity tube. Another 40 mice were inoculated with 50ul sterile saline nasal as the control group , All mice were anesthetized on 12 hours, 1 day, 3 days, 5 days and 7 days respectively. Bleed by removal of the eye, and dissect the mice.2. Killed the infection and control groups of mice on 12 hours, 1 day, 3 days, 5 days and 7 days respectively, and conduct the following studies:①Done fixed biopsy with the upper lobe of left lung, kidney, spleen, and small piece of liver.②Determinated the mouse lung tissue's TLR2, TLR4 and MyD88 mRNA expression levels in mouse lung tissue using the Real-time RT PCR .③Assay the cytokines (IL-6, TNF-α) in mouse lung homogenate by ELISA.3. SPSS16.0 statistical software was used for data entry and analysis.Results①Pathology results showed that, mice in infection group had inflammatory cell infiltration within interstitial of lung tissue, which increased with time. Walls of pulmonary capillary vessels haemorrhage suggested that modeling successfully. Mice in this group hemorrhaged in glomerulus, and had inflammatory cell within filtration. These mice also hemorrhaged in spleen medulla, and had neutrophil infiltration. There had not been pathological changes of liver in these mice.②The result of lung/body coefficient shows that there is statistical significance in the lung/body coefficient difference between infection group and control group(F=13.987,P<0.001), the difference of each timing shows statistical significance(F=3.035,P=0.023),the interaction difference of grouping and timing shows no statistical significance(F=1.232,P=0.305).There is statistical significance in the results of the difference of spleen/body coefficient between infection group and control group(F=40.585,P<0.001),the difference of each timing also shows statistical significance(F=4.525,P=0.003),and the interaction difference of grouping and timing shows no statistical significance(F=1.173,P=0.330). There is statistical significance in the results of the difference of kidney/body coefficient between infection group and control group(F=20.473,P<0.001),the difference of each timing also shows statistical significance(F=5.025,P=0.001),and the interaction difference of grouping and timing shows no statistical significance(F=1.131,P=0.274)There is no statistical significance in the results of the difference of heart/body coefficient between infection group and control group( F=0.061,P=0.805),the difference of each timing also shows no statistical significance(F=1.528,P=0.204),and the interaction difference of grouping and timing shows no statistical significance(F=1.144,P=0.343).There is no statistical significance in the results of the difference of liver/body coefficient between infection group and control group(F=0.590,P=0.478),the difference of each timing shows statistical significance(F=37.952,P<0.001),and the interaction difference of grouping and timing shows no statistical significance(F=2.303,P=0.067).There is no statistical significance in the results of the difference of brain/body coefficient between infection group and control group(F=0.031,P=0.861),the difference of each timing also shows no statistical significance(F=1.221,P=0.310),and the interaction difference of grouping and timing shows no statistical significance(F=0.355,P=0.840).③Compare with Relative expression of TLR2,TLR4,MyD88 mRNA in infection group and control group, the real-time RT-PCR testing result shows that there is no statistical significance of TLR2 mRNA expression between infection group and control group in Lung Tissue of mice (F=0.032,P=0.858), the difference of each timing also shows no statistical (F=0.068,P=0.991), and the interaction difference of grouping and timing shows no statistical significance(F=0.269,P=0.897).There is no statistical significance in expression difference between expression infection group and control group of TLR4 mRNA in Lung Tissue of mice (F=0.321,P=0.573), the difference of each timing also shows no statistical significance (F=0.443,P=0.777), and the interaction difference of grouping and timing shows no statistical significance(F=0.616,P=0.625).There is no statistical significance in expression difference between expression infection group and control group of MyD88mRNA in Lung Tissue of mice (F=0.711,P=0.402), the difference of each timing also shows no statistical significance(F=0.367,P=0.831), and the interaction difference of grouping and timing shows no statistical significance(F=1.031,P=0.398).④The difference of IL-6 concentrations expression between infection group and control group of Lung Tissue of mice shows statistical significance (F=69.18,P<0.001),the difference of each timing also shows statistical significance(F=5.289,P<0.001), and the interaction difference of grouping and timing shows statistical significance(F=8.686,P<0.001).The difference of TNF-αconcentrations expression between infection group and control group of Lung Tissue of mice shows statistical significance (F=24.6,P<0.001), the difference of each timing also shows statistical significance(F=14.230,P<0.001), and the interaction difference of grouping and timing shows statistical significance(F=13.987,P<0.001).Conclusion①Mouse intranasally inoculated by 50ul 1.2×109 /ml bacterial suspension of Legionella infection can establish Legionella model well.②This study has not yet found that TLR2, TLR4 and MyD88 involved in anti-Legionella infection. IL-6 and TNF-αwere related with Legionella infection. |
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