| ObjectiveTo establish the acute murine pulmonary tuberculosis model and evaluate the dynamic evolution of micro-CT (Micro-computer tomography) findings with the pathological changes in different periods of infection. According to the proteomic analysis of Mycobacterium tuberculosis (Mtb.) and mice at the different periods of infection in the lung tissue, we search for special proteins to be the molecular targets, which are for the preparation of monoclonal antibody targeted contrast agent of computed tomography.MethodsThe C57BL/6J mice were infected with standard strain of tubercle ballicus H37Rv by intranasal administration in ABSL-3,the healthy control groups were kept in SPF level lab. Micro-CT scans were undertaken at one, four, eight,12 weeks after infection, and then the mice were sacrificed. After tackled with heart perfusion, the lungs were harvested. About 1/4 of lung tissues were for Mycobacterium tuberculosis culture after homogenate; the second 1/4 were for proteomics analysis for screening of molecular marker of Mycobacterium tuberculosis and exploring immune mechanism of the host at different time following the infection respectively; the third 1/4 were stored in the -80℃ freezer for real time-PCR; and the last 1/4 lung tissue were for histopathology.According to the acute murine pulmonary tuberculosis model established, we analyzed the imaging findings of micro-CT corresponding to the pathological changes in different periods of evolution (the first part).In vivo proteomics of the tubercle bacillus were analyzed by shotgun liquid chromatography-mass spectrometry (shotgun LC-MS/MS). After processing of proteins isolated from lung tissue, mass spectrometry, searching the database of Mycobacterium tuberculosis, we got the Mtb proteome. Then analyse the overlapped proteins according to functional classification, and choose some protein, which may be cell membrane or cell wall protein, to be potential molecular target for targeted contrast agent.(the second part).Two-dimensional electrophoresis and liquid chromatography-mass spectrometry (2DE LC-MS/MS) were used in searching for differential proteins following the dynamic evolution of tuberculosis. After 2DE gel analysis, gel digestion, mass spectrometry, and searching database, we sreen the potent proteins as biomarkers Western Blot and RT-PCR were processed for validation (the third part).Results1.40 mice were infected. Nine of them died and excluded from the experiment groups. So the actual number of mice in group 1,2,3,4 were seven, six, eight, seven, respectively.2. Micro-CT-Pathology analyses:One week after infection, alveoli exudation in large area of lung, while micro-CT shows widely ground-glass opacities (GGO). Histopathologic changes show alveolar septal thickening with many lymphocytes and scattered neutrophils, macrophages and epithelioid cells. Four weeks after infection, at high magnification, a large number of giant macrophage and foam cells and some epithelioid cells, multinucleated giant cells were seen. Micro-CT showed patchy consolidation and GGO with ill-defined margin. Eight weeks after infection, the size of consolidation is enlarged, histopathologic changes show the number of epithelioid cells increased, and there is a tendency of tuberculosis nodules formation.12 weeks after infection, on one hand, there is little inflammation with scattered a couple of small tuberculosis nodules, on the other hand, there is more widely consolidation. Log 10 cfu of bacteria Load of lung at different infected time point is 6.30±0.02,6.61±0.40,6.38±0.25,4.52±0.32, respectively.3. Results of "shotgun" LC-MS/MS proteomics:the protein samples of experimental groups at four, eight,12 weeks after infection are identified 102,101 and 90 proteins respectively, and a total of 237 proteins. Protein expression overlap: a total number of 43, accounting for 18.2%. There are 11 overlapped proteins among 3 groups,12 overlapped proteins between 4 weeks and 8 weeks after infection,20 proteins beteewn 8 weeks and 12 weeks after infection and only 2 proteins between 4 weeks and 12 weeks after infection. Classify these proteins according to protein function. The largest category is class 7 (cell metabolic intermediates), the second categoty are class 2 (signal proteins) and class 9 (regulation proteins). According to the function of proteins and wether to be cell membrane or not, we choose the two-factor systems MtrA and MtrB which are taken much attention in recent years are chosen to be the potential biomarkers.After Western-Blot, we thought MtrB, as a membrane protein, may be used as a target for preparation of contrast agent.4. Molecular targets screening with 2-DE proteomics:Analysis four groups of 2D gels, at one, four, eight,12 weeks after infection, there are 11,154,254 and 30 differently expressed protein-points respectively. There is different overlap about the points between four groups, three groups and two groups, with 0,14 and 75 protein-points respectively.145 protein-points were chosen to process gel digestion. Researching the house mouse protein database with the MS/MS results,82 non-redundant proteins were accurately identified. Among these proteins the interacted proteins contain:Alb, Vim, Actb, Fthl, Tnnt3, Ftl, Msn and so on; and then comparing their difference ratio of the interacted proteins in different time points, we found that Fthl and Ftll were up regulated at four weeks, eight weeks and 12 weeks after infection, showing a parabolic pattern.Additionally, Researching the Mycobacteria tuboculosis protein database with the 2D MS/MS results, there were only seven non-redundant proteins containing SYA, CP126, HTPG, Y224, SMC, ACEA.Conclusion1. Acute pulmonary tuberculosis mouse model of C57BL/6J is successful established and verified by tubercle bacillus culture, micro-CT and histopathologic changes.2. Micro-CT findings in acute pulmonary tuberculosis in mice have some characteristics following the disease process. There is large area of GGOs at acute phase-different size of consolidations-large-scale consolidation and nodules with ill-defined margin (granulomas)-further progressive consolidations, or consolidations are partly absorbed with scattered smaller nodules. The micro-CT findings can reflect the histopathologic changes, be used to monitor the course of murine tuberculosis, evaluate the experimental process and reduce the sacrifice of mice in some experiments.3. MtrB (Rv3245c), as Mycobacterium tuberculosis membrane protein, can be a potential molecular target for CT contrast agent. The study provides the research basis for preparation of anti-MtrB monoclonal antibody target to CT contrast in animal models, which is just the research next. And it also provides an experimental basis in the specific diagnosis of early tuberculosis.4. Differential proteins expressed by the host are changing with the development of tuberculosis process. Fthl and Ftll may be used as a new biomarker of tuberculosis. The deep study of regulatory mechanisms of Fthl and Ftll in macrophages infected by Mycobacterium tuberculosis provides new ideas for the prevention and treatment of tuberculosis. |
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