The Study of the Gene Expression of HMGB1 in Peripheral Blood Mononuclear Cells of Lupus and Its Clinical Meaning
|School||Southern Medical University,|
|Course||Dermatology and Venereology|
|Keywords||Systemic Erythematosus High mobility group box protein 1 SLEDAI|
Background: Systemic Lupus Erythematosus (Systemic lupus erythematosus, SLE) is a multi-factors involved in autoantibody production and immune complex deposition and multi-system damage characteristic of autoimmune diseases. The pathogenesis of SLE has long rheumatism the immune research areas of difficulty. Therefore, to study the pathogenesis of SLE, finding effective treatment measures for improving the quality of life of patients, has a very important significance. Immune dysregulation plays a dominant role in the pathogenesis of SLE, including abnormal function of immune cells. Cytokines by immune cells and certain non-immune cells by stimulating synthesis and secretion of a class of biologically active substances. As a cell signaling molecule, mainly mediated regulation of immune response and inflammatory response, stimulate hematopoietic function, and to participate in tissue repair. SLE patients is the existence of multiple cytokines abnormal expression, in the course of the disease, there are significant cytokine network imbalance, cytokine profiles offset. With the development of molecular biology techniques in recent years and found that the degree of imbalance between the the SLE disease occurrence and development process of proinflammatory cytokines and anti-inflammatory cytokines determine the severity and scope. High mobility group box protein 1 (high mobility group protein box 1, HMGB1) is a nuclear protein involved in a variety of biological functions in the nucleus, the latest study found that it is a new kind of inflammatory cytokines. Recent studies have found that, HMGB1 expression in the cytoplasm, cell membrane, and can also be secreted into the extracellular. The mononuclear macrophages by inflammatory stimuli (LPS, TNFa, IL-1, etc.), can take the initiative to make nuclear HMGB1 transferred to the cytoplasm, nonclassical secretory pathway via protein secreted into the extracellular. Another necrotic cells can also be passive release of HMGB1. Extranuclear HMGB1 is an acute or chronic inflammation of proinflammatory cytokines can stimulate endothelial cells, the mononuclear macrophages secrete inflammatory cytokines and adhesion molecules, and recruitment of inflammatory cells to the site of injury, a large number of cytokines induced secretion mediated guide a variety of acute and chronic inflammatory diseases, such as septic shock, acute lung injury, cardiovascular disease and rheumatic autoimmune diseases, HMGB1 is an endogenous immune adjuvant, dendritic cell maturation and Th1 cells polarization process of an adjustment factor, HMGB1 outside the cell can be induced TNF-a, IL-1, IL-6, and a variety of adhesion molecules such as of VCAM-1 and MCP-1 expression, while HMGB1 for maintaining Th1/Th2 the balance between cytokine also plays an important role. Previous research has confirmed, HMGB1 and various autoimmune diseases, and has become a target for arthritis treatment. But whether to play a role in the pathogenesis of SLE process and its possible mechanism of action is unclear, though there are already a small number of studies have reported about HMGB1 and the pathogenesis of SLE relations abroad, but the results are different. Therefore, this study intends to clinical data and laboratory detection using a more mature ELISA method to detect the plasma of patients with] (?) MGB1 levels, and plasma HMGB1 concentration and SLE disease activity indicators between correlation analysis to study the role of HMGB1 in the pathogenesis of SLE and observed PBMCs the HMGB1-mRNA gene expression changes, and to explore the possible role of HMGB1 in SLE disease mechanisms and new therapeutic strategies and experimental treatment of SLE basis. Purposes: 1. Detected in patients with SLE plasma HMGB1 levels of HMGB1-mRNA expression in peripheral blood mononuclear cells and to explore the possible role of HMGB1 in SLE disease mechanisms. 2 by analyzing patients with SLE plasma HMGB1 levels and peripheral blood mononuclear cells] (?) MGB1-mRNA expression level SLEDAI and other indicators of disease activity explore the association of HMGB1 with SLE disease activity, as judged SLE prognosis provide a theoretical basis, and at the same time provide new therapeutic strategies for the treatment of SLE. Method: 1, patient characteristics selected Nanfang Hospital of Southern Medical University skin rheumatology outpatient and hospitalization in patients with SLE, 44 cases are in line with the 1997 American Rheumatism Association (American College of Rheumatology ACR) revised the diagnostic criteria for classification of SLE. Including 26 cases of active SLE, SLE stable group of 18 patients, 18 cases of the sex and age of the 44 patients matched healthy volunteers as normal control group. All into the group of people there is no history of smoking and combined oncology and infectious diseases, patients over and volunteer their consent and signed an informed consent form. All patients were detected in the blood, liver, kidney function, autoantibodies, humoral immunity, CRP, ESR, IgG, a 24-hour urine protein record clinical and laboratory parameters. 2, HMGB1 expression levels detected in the peripheral blood of patients with systemic lupus erythematosus, take the 44 SLE patients and 18 healthy volunteers crowd fasting peripheral blood plasma using enzyme-linked immunosorbent assay (Enzymelinked immunosorbent assay, ELISA) serum The concentration of the HMGB1. Operating procedures are summarized as follows: 1 plus samples and standards, coupled with biotin-labeled antibody, 37 ℃ reaction, 60min; (2) wash solution was washed 4 times; ③ plus pro and streptavidin-HRP, 37 ℃ incubated for 30min; ④ washing solution was washed four times; ⑤ add substrates A and B, 37 ° C incubated for 10min; ⑥ The stop solution, using a microplate reader 450nm readings. 3 patients with systemic lupus erythematosus HMGB1 mRNA expression in peripheral blood mononuclear cells select the 44 SLE patients and 18 healthy volunteers crowd the peripheral blood of 10 ml of fasting venous detected by reverse transcription - polymerase chain reaction (reverse transcriptase -polymerse chain reaction, RT-PCR) detection of peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) expression of in HMGB1mRNA in the case. RT-PCR procedure is as follows: ① density gradient separation separation of peripheral blood mononuclear cells, plus TRIZOL cracking cells: ② TRIZOL method of total RNA extracted mononuclear cells; the ③ gel electrophoresis observe the integrity of RNA, UV spectrophotometry the meter detects RNA concentration; ④ under genebankHMGB1 gene sequences using the cDNA was synthesized using the total RNA extracted template primer5.0 Design and Synthesis of HMGB1, β-actin primer sequences; ⑤; ⑥ cDNA sequence amplification template, HMGB1, β-actin primers; ⑦ gel electrophoresis, gel imaging analysis system for grayscale scanning, the results in order to the grayscale of HMGB1/β-actin than that; ⑧ PCR product of gene sequencing. Statistically: all data input SPSS13.0 package, two sets of samples between the mean compare the use of two independent samples t-test comparison between the mean between multiple samples meet the homogeneity of variance, using one-way analysis of variance (One-way ANOVA) and the number of samples between multiple comparisons (LSD method); does not satisfy the homogeneity of variance, using approximate F-test (Welch Act) and the correction for multiple comparisons (Dunnett's T3 method); relationship between the dual variables using bivariate correlation analysis; Fitting Curves Curve Estimation and select the best curve fit equation based on curve fitting of the coefficient of determination R2; selected a = 0.05 as the level of inspection results data x ± SD. Results: (1) patients with active SLE plasma HMGB1 levels (363.44 ± 302.226ng/ml) was significantly higher than the normal control group (110.178 ± 100.078ng/ml) (P lt; 0.01) and patients with active SLE group (142.75 ± 153.811 ng/m1) (P lt; 0.05); were no significant differences between patients with active SLE group and the normal control group. HMGB1 mRNA (2) patients with active SLE group relative expression level (3.493 ± 2.825) than in patients with active SLE group (1.785 ± 0.735) (P lt; 0.01) and normal control group (1.179 ± 0.792) (P lt; 0.05), rather than the group of patients with active SLE HMGB1 mRNA relative expression level of the normal control group showed no significant difference. (3) SLE patient group plasma HMGB1 levels with SLEDAI, ESR, C3, ds-DNA, a 24-hour urine protein was positively correlated; the plasma HMGB1 protein expression levels of C4, ALB was negatively correlated. SLE patients PBMC HMGB1-mRNA expression and SLEDAI and plasma HMGB1 levels were positively correlated. Conclusion: Our results show that HMGB1 may not be involved in the early pathogenesis of SLE, but when the development of the disease to a certain extent, HMGB1 secretion increased, may play an important role in the further development of this disease, suggesting that HMGB1 SLE pathogenesis important pro-inflammatory cytokines, one might become a sensitive indicator of judgment SLE clinical condition changes. We further use of the RT-PCR method to detect the expression levels of HMGB1-mRNA in PBMCs of 44 SLE patients and 18 normal controls, confirmed the above findings, suggesting that the gene level HMGB1 in SLE disease progression from an important role in the plasma levels of HMGB1 and HMGB1-mRNA expression levels can be used as a judgment SLE disease activity and severity indicators. 3. Speculated HMGB1 in SLE immune function may be pre-inflammatory cytokines in patients PBMC surface receptor binding to inspire some sort of transmembrane signal transduction pathway to signal incoming nucleus, causing the expression of the target gene HMGB1, take the initiative to synthetic secrete HMGB1, the peripheral blood of patients the plasma HMGB1 expression levels were significantly increased, further pathogenic cytokine network disorder. The intervention of the activity of HMGB1 may become a new therapeutic strategy for SLE. HMGB1 signal transduction mechanism further clarify, HMGB1 drugs that target is expected to become a new therapeutic approach for the treatment of SLE. In summary, the experiments detect two aspects from the clinical data and laboratory research results show: HMGB1 plays an important role in the pathogenesis of SLE, is also a concern in the treatment of SLE new molecular therapeutic target point. However, this study only in respect of its expression in the peripheral blood plasma and mononuclear cells in SLE patients studied experimental animal models, needs the help of in-depth study and discussion of its role path.