Preliminary Study On The Founction Of HMGB1 In RA Patients

Objective:To clone and express mouse HMGB1 gene and prepare the rabbit polyclonal antibody against HMGB1; to detect the expression levels of high mobility group box chromosomal protein 1 (HMGB1) and Th17 cells transcription factors, related cytokines in peripheral blood in rheumatoid arthritis (RA) patients and analysis the relations of them in RA patients. For the first time, we investigate the feasibility of HMGB1 in RA patients based on the founction of rHMGB1 to induce Th17 in vitro.Method:1) HMGB1 gene was cloned from spleen cells of mouse by RT-PCR, The HMGB1 gene was constructed into pMD18-T vector. The plasmid was transformed into E.coli DH5αand identified by single and double restriction enzyme as well as sequencing.2) HMGB1 gene was constructed into pET28 vector and expressed in E. coli. Rossta to get fusion HMGB1 protein about 30KD with His tag; The fusion protein was purified by IMAC Ni-Charged Resin column of Bio-Rad company; The purified protein was tested with anti-His by Western-blot. The rabbit was immunized by the target fusion protein. Then the blood serum containing HMGB1 polyclonal antibody was collected. The titer of antibody was confirmed by enzyme-linked immunosorbent assay (ELISA) .3) All samples of peripheral blood mononuclear cells including 80 patients were collected from Jiangsu University affiliated hospital -Zhenjiang First Hospital. Including 32 rheumatoid arthritis (RA) patients in unactive phase and 48 patients in active phase, and 50 healthy volunteers.4) The mouse spleen CD4~+Tcells were stimulated by HMGB1.5) The related genes&proteins of HMGB1 and Th17 cells expression levels were detected by quantitative realtime-PCR (QRT- PCR) and enzyme linked immunosorbent assay (ELISA) from PBMC of samples.6) The ratio of CD3~+CD8IL-17~+cells in PBMC were detected by FACS.Results:1. The results of clone, expression and polyclonal antibody of mouse HMGB1.1) The full length of HMGB1 gene was cloned successfully from human PBMC and shared 100% concordance with the sequence of mouse HMGB1 in Genebank.2) The HMGB1 fusion protein was expressed in E. coli. and purified. The fusion was about 30KD. 3) We prepared the polyclonal antibody against mouse HMGB1, which was approved with high reactivity by ELISA, respectively. And it was approved that HMGB1 protein had high immunogenicity.2. The results of rheumatoid arthritis patients related detections1) The results of QRT-PCR showed: Compared with healthy volunteers, RA patients have higher mRNA levels of HMGB1 and Th17 master transcription factor RORγt and related cytokines IL-17 mRNA in the PBMCs (P<0.05) ; especially in active RA patients compared with unactive phase patients (P<0.05) . Positive correlations were found between the expression levels of HMGB1 and Thl7-associated factors in RA patients.2) The results of ELISA showed: Compared with healthy volunteers, RA patients have higher levels of IL-17, IL-23 (P<0.05 ) .3) The results of FACS showed: Compared with healthy volunteers, RA patients have higher levels ratio of CD3~+CD8~-IL17~+T cells.3. The results of experiment in vitroThe results of QRT-PCR of mouse CD4~+T cells were cocultured with mouse HMGB1 protein showed: Time-dose effective was existed in the role of HMGB1 induction Th17. When the concentration of HMGB1 reach at 100ng/ml, and coculture with CD4~+T cells 12h had the best result in inducing Th17 cells. Conclusions:HMGB1 gene was cloned and expressed in E.coli system effectively; The purified protein was obtained and the the anti-HMGB1 polyclonal antibody was prepared successfully; HMGB1 and Th17 cells related factors expression in PBMC with RA patients were obviously higher than healthy control groups and there was a positive correlation between the expression of HMGB1, Th17 cells related factors and the related clinical experiments in RA patients, which indicated that HMGB1 may be related to the RA patients. HMGB1 was helpful to the differentiation of Th17 cells in vitro, as the tissue injury signals, except incurred macrophages play a role in tissue damage localization in early anti-infection, in the process of the chronic immune and inflammatory damage, also constitutes RA pathology part of the process, through the regulation of immune cells to participate and increase the inflammatory injury. It will be the basis for studying the role of HMGB1 in pathomechanism of rheumatoid arthritis and the treatment based on HMGB1.