| Objective: By observing the expression changes of IL-35 and iTr35 cell subsets after chronic infection of Mycobacterium tuberculosis,the immune effect in infection and the possible immune mechanism of infection were investigated.Methods:(1)C57BL/6 male mice were infected with H37 Ra M.tb strain by tail vein injection to produce M.tb infection model;(2)Mouse left lung was homogenized and then inoculated.On the Roche medium,the bacteria counted when the colonies were visible to the naked eye;(3)the lungs were taken for pathological tissue sectioning and staining to observe the lesions;(4)Flow cytometry was used to detect the spleen cells of each group of mice.The proportion and changes of iTr35 cell subsets were observed;(5)Real-time quantitative PCR was used to detect the expression and change of IL-35 mRNA in spleen cells of control group and each infected group;(6)Detection by Westen Blot method The expression and changes of IL-35 protein in various mouse spleens were observed.(7)The expression levels and changes of IL-35 in serum of each group were detected by ELISA.Results: 1.Comparison of EBI3 mRNA expression levels in each group: Compared with the control group,the expression level of EBI3 mRNA in the infected group was significantly increased(0.873±0.11 vs.1.600±0.23 vs.4.638±1.63,P<0.01),including infection for 4 weeks.The expression level of EBI3 mRNA in the group was not significantly different from that in the normal control group(P>0.05),while the expression in the 8-week group was higher than that in the normal control group and the 4-week infection group(P<0.01).The expression levels of p35 mRNA in each group were significantly higher than those in the control group(0.724±0.38 vs.1.792±0.29 vs.4.744±0.78,P<0.01),and the infection group was 4 weeks.The expression level was higher than that of the normal group(P<0.01),and the expression level of the infection group was higher than that of the infection group for 4 weeks(P<0.01).2.The relative expression of EBI3 protein in each group was significantly higher than that in the control group(0.441±0.03 vs.2.154±0.36 vs.2.955±0.06,P<0.01).The expression level of EBI3 protein in the 4 weeks group was significantly higher than that in the normal control group(P<0.01),while the expression of EBI3 protein in the 8 weeks group was higher than that in the normal control group and the 4 weeks infection group(P<0.01).3.The expression levels of p35 protein in each group were significantly higher than those in the control group(0.251±0.01 vs.2.078±0.16 vs.2.641±0.03,P<0.01),and the infection group was 4 weeks.The expression level of p35 protein was higher than that of the normal group(P<0.01).The expression of p35 protein in the 8 weeks of infection was higher than that in the 4 weeks of infection(P<0.01).Compared with the control group,the serum IL-35 expression level of the infected group was significantly increased(23.726±3.21 vs.46.069±3.10 vs.61.494±6.23,P<0.01),and the serum IL-35 was infected for 4 weeks.The expression level was significantly higher than that of the normal control group(P<0.01),while the expression level of IL-35 was higher in the 8-week group than in the normal control group and the 4-week infection group(P<0.01).4.Compared with the normal group of mice,the proportion of iTr35 cell subsets in the infected model group increased(84.34±3.67 vs.87.40±1.32 vs.90.52±2.15,P<0.01);iTr35 cells were infected in the 4 week model group.The proportion of subgroups was higher than that of the normal group,but it was not statistically significant(P>0.05).The proportion of iTr35 cells in the model group was higher than that in the 4 weeks group,but it was not statistically significant(P> 0.05).Conclusion: 1.The mice in the experimental group were injected with the H37 Ra attenuated M.tb strain in the tail vein,and the chronic M.tb infection model was successfully replicated,which laid the experimental foundation for the infection of M.tb infection.2.The expression of IL-35 in the spleen and peripheral blood of mice infected with M.tb is increased,and infected 8W group was more obvious than the infected 4W group,suggesting that IL-35 expression may play an important immune function in chronic infection of M.tb.3.The proportion of iTr35 cells in the spleen of mice infected with M.tb increased,and the increase was more obvious when infected with 8W,suggesting that iTr35 cell subset differentiation may play an important role in the chronic infection of M.tb adjustment function. |
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