| Objective: To detect the difference of Tollip and TLR4 expression in macrophage by immunofluorescence technique,flow cytometry,RT-PCR and real-time fluorescent quantitative PCR in peripheral venous blood of patients with osteoarticular tuberculosis.To explore the mechanism of action of Tollip and TLR4 expression differences in patients with osteoarticular tuberculosis.Methods: Thirty patients with osteoarticular tuberculosis(11 cases of thoracic tuberculosis,14 cases of lumbar tuberculosis,5 cases of knee joint tuberculosis)were enrolled in the hospital affiliated to Youjiang Medical University for Nationalities and Baise City People’s Hospital from October 2017 to January 2019);In the same period,30 normal people(normal group)who had a physical examination at the Physical Examination Center of the Affiliated Hospital of Youjiang Medical College for Nationalities and Baise City People’s Hospital were selected.The expression levels of Tollip protein and TLR4 protein were detected by immunofluorescence assay,the expression levels of Tollip protein and TLR4 protein were detected by flow cytometry,and the expressions of Tollip mRNA and TLR4 mRNA in macrophages were detected by real-time fluorescent quantitative PCR.The difference in expression between the two groups was compared.Results: The results of TLR4 mRNA and Tollip mRNA were determined by RT-PCR and real-time fluorescent quantitative PCR in the osteoarticular tuberculosis group: The relative expression of TLR4 mRNA was 21.537±0.823,and TLR4 ΔCt was 10.622±1.447.The relative expression of Tollip mRNA was 21.453±1.211 on average,and Tollip mRNA ΔCt was 10.694±1.391.The health group showed that the relative expression of TLR4 mRNA was 21.457±0.470,the TLR4 ΔCt was 10.617±1.389,the relative expression of Tollip mRNA was 21.573±0.707,and Tollip mRNA ΔCt was 10.452±1.546,The values of the bone joint tuberculosis group and the healthy body test group were calculated as TLR4 average t=0.462,TLR4ΔCt t=0.014,Tollip average t=0.477,Tollip ΔCt t=0.637.TLR4 average P=0.646,TLR4ΔCt t=0.989,Tollip The average t=0.635,Tollip ΔCt t=0.527,and the P values were all >0.05,which was not statistically significant..The expression of TLR4 protein and the positive rate of Tollip protein were detected by flow cytometry in osteoarticular tuberculosis group: TLR4 was 1.40±0.63 and Tollip was 5.38 ± 1.99.The healthy group was: TLR4 was 0.40 ± 0.26,Tollip was 1.40 ± 0.53,TLR4 positive rate t was-10.588,and Tollip positive rate t was-8.075.The positive rate of TLR4 was 0.000,and the positive rate of Tollip was 0.000,both less than 0.05.The positive rate of TLR4 protein and Tollip protein in the osteoarticular tuberculosis group was significantly higher than that in the healthy group,with statistical significance P<0.05.The expression of TLR4 protein and Tollip protein in osteoarticular tuberculosis group was detected by cellular immunofluorescence technique.It was found that the number of TLR4 protein in the osteoarticular tuberculosis group was higher than that in the healthy group,and the brightness was significantly higher than that in the healthy group.The Tollip protein in the osteoarticular tuberculosis group was larger than that in the healthy group,and the brightness was also brighter.It was also found that the blue fluorescent brightness of the Tollip protein region in the macrophage of TLR4 protein with high brightness red fluorescence in the osteoarticular tuberculosis group was significantly darker,while the blue fluorescence intensity of the Tollip protein region in the dark fluorescent red fluorescent macrophages of TLR4 protein was significantly brighter.Conclusion: This study revealed that TLR4 protein and Tollip protein expression can be increased in patients osteoarticular tuberculosis after tuberculosis infection.This study preliminarily explored the excessive abnormal expression of TLR4 on the surface of macrophage in peripheral venous blood of patients with osteoarticular tuberculosis and may have the relationship of Tollip negative feedback regulation,but its specific mechanism of action has not been clarified. |
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