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Effects of high mobility group box-1 protein on cytokine expreesion in splenic dendritic cells in rats / 中华急诊医学杂志
Chinese Journal of Emergency Medicine ; (12): 127-131, 2009.
Article in Chinese | WPRIM | ID: wpr-396769
ABSTRACT
Objective To investigate the effect of high mobility group box-1 protein (HMGB1) on cytokine expression in splenic dendritic cell (DCs). Method DCs isolated from the spleens of male Wistar rats were seed-ed on 96-well (1×10s cells/well) cell culture plates, and the cells were stimulated with HMGB1 for various length of time or in different concentrations. (1) The time-dependent response between HMGB1 and tumor necrosis factor-α(TNF-α) as well as interleukin-12 (IL-12) gene/protein expressions 24 wells of DCs were dividedly into six groups including 24 h-normal controls (n=4), 48 h-normal controls (n=4), 72 h-normal controls (n=4), and 24 h-HMGB1 treated group (n=4), 48 h-HMGB1 treated group (n=4) as well as 72 h-HMGB1 treated group (n=4), respectively. Among three HMGB1-treated groups, DCs were stiraulated by 1 μg/mL HMGB1. DCs were denatured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvested to determine Il-12 as well as TNF-α protein levels. (2) The dose-dependent response between HMGB1 and TNF-α as well as IL-12 gene/protein expressions 16 wells of DCs were dividedly into four groups in-cludingnormal controls (n=4), 0.1 μg/mL HMGB1 treated group (n=4), 1 μg/mL HMGB1 treated group (n =4), and 10 μg/mL HMGB1 treated group (n=4), respectively. After stimulated for 48 h, DCs were dena-tured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvest-ed to determine IL-12 as well as TNF-α protein levels. Total RNA was extracted from cells using the single-step technique of acid guanidinium thiocyanate-chloroform extraction according to the manufacturer' s instruction (Promega, Madison, WI). mRNA for TNF-α and IL-12 were quantified by SYBR Green two-step, real-time re-verse transeription-polymerase chain reaction taking glyceraldehyde-3-phesphate dehydrogenase (GAPDH) as an internal standard. Levels of IL-12 and TNF-α in cell culture supernatants were determined with ELISA, strictly fol-lowing the protocols provided by the manufacturer. Data were analyzed with a one-way ANOVA. A P-values <0.05 were considered statistically significant. Results After stimulation with 1 μg/mL HMGB1, IL-12 and TNF-α protein and gene expressions in rat splenic DCs were markedly up-regulated at 24 h to 72 h (P<0.05 or P<0.01), and the expression levels of IL-12 and TNF-α peaked at 48 h (P<0.01). When DCs were cultured in the presence of 0.1 μg/mL, 1 μg/mL, and 10 μg/ml HMGBI for 48 h, expressions of IL-12 and TNF-α were also significantly up-regulated (P<0.01), and values of these cytokines were highest in 1 μg/mL HMGB1-treated group (P<0.01). Conclusions These data suggest that HMGB1 appears to be a potential immunostimulatory signal that induced DC maturation, and HMGB1 stimulation can result in marked up-regulation of IL-12 as well as TNF-α synthesis and release in splenic DCs.

Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Chinese Journal of Emergency Medicine Year: 2009 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Chinese Journal of Emergency Medicine Year: 2009 Type: Article