Macrophages Enhancing Tuberculosis-Mycobacterium-Eliminating Effect Research

Objective:Autophagy is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. It is a tightly-regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products.It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes.This research is aimed to justify the 3-methyl-adenine's(3-MA)and the rapamycin's effect in adjusting macrophages'autophagy activity and helping to eliminating the tuberculosis mycobacterium through some experiments including the tests on mice's original macrophages and living mice.Methods:The research is conducted in two steps:in-vitro experiment and in-vivo experiment. Step 1:in-vitro experiment. Lavage the healthy mice's lungs to get the original macrophages, and then randomly divide these macrophages into four groups:the control group, the inhibition group, the IFN-y group, and the induced group.All groups are firstly cultivated in the fetal bovine serum DMEM medium for two days,then separately added in with 3-MA 60 mg/L,IFN-y 400U, or rapamycin 50nmol/l, and some time later, separately added in with tuberculosis mycobacterium strain H37Rv 1×10^7 CFU/L,and after three-hour cultivation period, all groups are flushed thrice with the PBS pH7.4 buffer solution to remove the non-combined H37Rv strain. Conduct the fluorescent quantitative PCR of the quantified flushing liquid to measure the remaining tuberculosis mycobacterium DNA after the macrophages'autophagy effect, finding the macrophages'autophagy effect difference between these four groups.Step 2:in-vivo experiment. Randomly divide the BALB/C mice (six-week-age) into four groups, and conduct the tuberculosis-mycobacterium-atomizing-infection to these mice, then do the intraperitoneal injection separately with 3-MA, IFN-y, or rapamycin (mice in the control group are injected with nothing).Seven weeks later, kill the mice through displacing cervical spines, and take pulmonary tissue slices under aseptic circumstance, do the HE staining or acid-fast staining to these slices, and then perform the tuberculosis mycobacterium colony counting to find the elimination effect of the tuberculosis mycobacterium in vivo.Results:1.Measure the tuberculosis mycobacterium numbers in the four groups with the quantitative real-time PCR method after the stimulated macrophages'autophagy effect, and the results show that the tuberculosis mycobacterium number of the IFN-y group is obviously lower than those of the other three groups.And the differences are statistically significant; 2.The two staining results of pulmonary tissue slices both show that the pulmonary tissue slices'pathological changes of the IFN-y group and the induced group are significantly lighter than those of the control group and the inhibition group;3.The tuberculosis mycobacterium colony counting of the pulmonary tissue serums shows that colony counts of the IFN-y group and the induced group are the lowest, and the differences are statistically significant.Conclusions:1.This research validates that the 3-methyl-adenine can restrain the macrophages'autophagy effect of tuberculosis mycobacterium, while the rapamycin can induce the macrophages'autophagy effect.2.This research validates that the rapamycin can enhance the macrophages'autophagy effect and IFN-y can induce the cellular immunity, and their effect in eliminating the tuberculosis mycobacterium are the same.