Differential Expression Of Proteins In Peripheral Blood Mononuclear Cells From Patients With Pulmonary Tuberculosis

Background and Objective:Tuberculosis (TB) is a chronic bacterial infection caused by a germ called Mycobacterium tuberculosis. The bacteria can damage any parts of the body, especially attack the lungs which calls pulmonary tuberculosis (PTB). At present, the incidence of TB is increasing gradually accord to the epidemic of HIV infection and drug resistant strain, immigration and poverty-striken. It is a major infection which threaten human health severely. It becomes a public health and social problem which is payed closer attention to all over the world.In the human cell mediated immunity, mononuclear macrophage is not only the major parasitizing cell of Mycobacterium tuberculosis, but also the major effector cell of host angtigen presentation and antibiosis. And lymphocyte is the major immunoregulation cell of the cell mediated immunity. So studying the peripheral blood mononuclear cells is important in understanding the immunologic mechanism of tuberculosis.Recently proteomics techniques provide powerful tools for studying molecular mechanisms and functions of key proteins involving in many diseases. Proteomics are to improved elucidate molecular classification of diseases and to discover sensitive biomarkers that are useful for diagnosing, treating, and predicting the prognosis of deseases. To our knowledge, no studies have been performed comparing proteomic profiles in peripheral blood mononuclear cell (PBMC) from patients with PTB. Therefore, this study we performed to identify the differentially expressed proteins in PBMC from PTB and health volunteers by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), aim to find out the key proteins involved in TB development, even to provide possible specific biomarker and establishing further targeted therapy for TB.Methods:(1)10 PTB and 10 health volunteers were enrolled in our experiment. PBMCs were collected by density gradient centrifugation. The total protein of PBMC were dissolved in upgraded cell disruption buffer and the protein concentration were measured by Bradford kits.(2) Two-dimensional electrophoresis (2-DE) was used to separate the total protein of PBMC from 10 PTB and 10 health volunteers. Images of CBB R520-stained 2D gel were alanyzed by ImageMaster 2D Elite 6.0 software through identifying spot, adjusting background, standardizing and matching, establishing matchset gel. The differential expression proteins were identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and searched in database of proteins by Mascot software. (3) The method of Western blot was used to determine the differential expressional levels of partial proteins.Results:(1) For PTB PBMC, average spots of 2 gels were 1103±25, with an average matching rate of 87.9%. For control, average spots of 2 gels were 1053±4, with a average matching rate of 86.3%. The average position deviation of matched spots was (1.17±0.18) mm in IEF direction, and (1.09±0.14) mm in SDS-PAGE direction. The well-resolved and reproducible 2-D gel maps of PBMC from PTB and normal controls were obtained (17cm PH3-10 NL IPG gel).(2) A total of 886±17 spots were matched between the electrophoretic maps of PBMC in 10 PTB and 10 normal controls. 14 differential protein spots were found and 11 of them were up expressed in PTB team. 12 proteins were analyzed and identified by PMF. Some proteins were related to the pathogenesis of PTB, such as Pleckstrin and Coronin-1A.(3) In order to validate the reliability of the identified results, the expression of Pleckstrin and Coronin-1A were detected by Western blot. The result displayed that Pleckstrin was down-regualted in PBMC of PTB, whereas it was up-regualted in normal controls, which was consistent with our 2-DE analysis results. There was no significant statistic association Coronin-1A expression.Conclusions:In this experiment, study of differential protein expression in PBMC from patients with pulmonary tuberculosis by comparative proteomics established the well-resolved, reproducible 2-DE patterns of PBMC from PTB and health volunteers and identified 12 different proteins. It suggests that Pleckstrin and Coronin-1A may have an important role in PTB pathogenesis through regulating the phagocytosis of mononuclear macrophage based on literature retrieval. Western blot confirmed that Pleckstrin was decreased expression in PTB. These results will provide a useful clue to elucidate the pulmonary tuberculosis pathogenesis.