The Study Of Immunologic Property For Severe Pulmonary Tuberculosis

Objective1. To investigate the gene expression differences in severe cases and mild cases of pulmonary tuberculosis by transcriptomics technology, and find the relationship between differential expression genes and the disease.2. To study the immune responses and immunopathologic mechanisms of severe pulmonary tuberculosis and to shed light on the molecular basis for the immune responses in TB patients and find therapy method of tuberculosis.Method1. The criteria were defined for patient selection and exclusion.2. Peripheral blood samples were collected from severe and mild patients with pulmonary tuberculosis at Institution of Tuberculosis Research of PLA 309th Hospital, and from healthy controls. PBMC were isolated and lysed with Trizol. Transcriptomics analysis was performed with Affymetrix Gene expression chips by CapitalBio Corporation in Beijing.3. Bioinformatics analysis were performed according the result of gene chip detection By using Cluster 3.0 for hierarchical cluster analysis. SAM (Significance Analysis of Microarrays) for differential gene expression, and MAS (Molecule Annotation System) for GO and pathway analysis.4. The results of gene expression profile were further verified by using real-time PCR (RT-PCR) or ELISA in TB patients and controls recruited according to the criteria used for patients selection5. ANOVA were used for homogenity test of variance and non-parametric tests for statistic analysis among the groups.Result1. Cluster analysis show that there were distinct gene expression profiles among severe and mild TB patients, and healthy controls.2. The criteria used for selection of differential expression genes by SAM was p<0.05, ratio ≥2 or ≤0.5. Compared with healthy controls, only 12 genes were down-regulated in patients with mild pulmonary TB. In contrast, patients with severe pulmonary TB had 317 up-regulated genes and 350 down-regulated genes compared with healthy controls. Comparison of patients with severe vs mild TB showed that 142 genes were up-regulated and 263 genes were down-regulated.3. MAS and Gene Ontology analysis showed that differential expression genes mainly involved biological process. Pathway analysis showed that differential expression genes of severe patients involved 35 significant pathways, that functions in immune respons, inflammation, tissue damage and remodling, apoptosis, anti-infection, metabolism and so on.4. The expression of selected genes was verified by RT-PCR and ELISA. Our RT-PCR analysis confirmed that CCL3, IL-1β, c-Jun, CXCL2 were down-regulated (p<0.05) in patients with severe pulmonary TB. Protein expression analysis by ELISA showed that patients with severe pulmonary TB had reduced expression for CCL4, IL-1β and c-Jun (p<0.05).ConclusionPatients with severe pulmonary TB exibited large number of differential expressed genes compared with healthy controls and patients with mild TB. These differential expressed genes involves complicated pathway network of immue response and inflammation, which might related to severe lung tissue damages in pulmonary tuberculosis.