The Effect Of TLR2 And TLR6 On Macobacterium Avium-Induced Apoptosis

Objective: Observing the changes of the expression of the crucial receptor and the secretion of the key cellular cytokines in different time period when macrophage was infected by mycobacterium avium to elucidate the mechanism about how mycobacterium avium survived inside macrophage.Also by blocking the regular expression of toll-like receptor 2 and toll-like receptor 6 on the surface of macrophage,analyzing the changes of amount of the cytokine TNF-alpha related to cell apoptosis,the changes of expression of the key apoptotic protein bax and bcl-2 and the changes of rate of apoptosis to elucidate the relationship between two key receptors and cell apoptosis.Method: The macrophage was infected by mycobacterium avium to build a mycobacterium avium infection model,the time period was settled as 4 hours,8hours,16 hours,24 hours,48 hours.Extracting total protein of cells and using western blotting to analyzing the changes of protein expression of crucial receptor Toll-like receptor 2 and Toll-like receptor 6 in different time period.And also extracting extracellular supernatant and using ELISA to analyzing the changes of key cellular cytokine TNF-?in different time period.Also by building a model of mycobacterium avium infection,to separate this experiment into five groups by adding the monoclonal antibody of toll-like receptor 2 and toll-likereceptor 6 to suppress the regular expression on the surface of macrophage:group1,macrophage without anything;group 2,macrophage infected with MA,the infection group;group 3,the infection group added with monoclonal antibody of TLR2;group 4,the infection group added with monoclonal antibody of TLR6;group 5,the infection group added with monoclonal antibody of both receptors.Then using ELISA to detect the expression of key cell cytokine TNF-alpha,using WB to analyzing the expression of apoptotic proteins named bax and bcl-2,at last,measuring the apoptosis rate of each group with the technology of flow cytometry.Result: The amount of TLR2 and TLR6 and the secretion of TNF-? was increased by time from 4 hours to 24 hours,and the amount of two receptors and the secretion of cytokines was somehow decreased by time from 24 hour to 48 hour.In the experiment of analyzing the relationship between apoptosis and these two receptors,the amount of TNF-alpha was decreased in group 3 and group 4 compared to group 2,and the amount was also decreased in group 5compared to group 3 and 4;the expression of bax was decreased in group 3 and group 4 compared to group 2,and the expression was also decreased in group 5compared to group 3 and 4;the expression of bcl-2 was increased in group 3 and group 4 compared to group 2,and the expression was also increased in group 5compared to group 3 and 4;when measuring the apoptosis rate of each group,we found the rate was decreased in group 3 and group 4 compared to group 2,and the apoptosis rate was also decreased in group 5 compared to group 3,4.Conclusion: These results have suggested that TLR2 and TLR6 was correlated with apoptosis of macrophages,mycobacterium avium could have influence on the apoptosis signaling pathway by affecting the normal expression of TLR2 and TLR6.