AIM: To investigate the expression of toll-like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells (KCs)and their role in ischemia-reperfusion (IR) injury of ratliver graft.METHODS: KCs were isolated at 0 (co...AIM: To investigate the expression of toll-like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells (KCs)and their role in ischemia-reperfusion (IR) injury of ratliver graft.METHODS: KCs were isolated at 0 (control group), 2, 12,24 h (IR group) following IR in rat liver graft, mRNA expressionof TLR4 and MD-2 was detected by RT-PCR analysis, proteinexpression of TLR4/MD-2 was detected by flow cytometric(FCM) analysis, and tumor necrosis factor-(~ (TNF-(~) levelin supernatant was measured by ELISA. Then isolated KCswere incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-(~ level was measured again.RESULTS: The mRNA and protein expression of TLR4/MD-2 and the level of TNF-(~ in IR group increased significantlyat 2 h following IR, and reached the maximum at 12 h, andslightly decreased at 24 h, but were still significantly higherthan those in the control group (P<O.01). The expression ofthese factors was markedly decreased after anti-TLR4antibody treatment as compared with the IR group (P<O.01).CONCLUSION: Lipopolysaccharide (LPS) following IR canup-regulate TLR4/MD-2 gene and protein expression inKCs, and synthesize cytokine TNF-(~. Anti TLR4 antibodycan inhibit the production of TNF-(~ induced by LPS. TLR4and its partner molecule MD-2 may play an important rolein Kupffer cell activation and IR injury.展开更多
Expression of Toll-like receptors at the maternal-fetal interface during normal and complicated pregnancies has aroused interest in the last few years. However, despite the importance of TLR-2 and TLR-4, which recogni...Expression of Toll-like receptors at the maternal-fetal interface during normal and complicated pregnancies has aroused interest in the last few years. However, despite the importance of TLR-2 and TLR-4, which recognizes most microorganisms presenting in the amniotic cavity infections associated with prematurity, comparison of the expressions of these receptors is rare in the literature. Thus, the purpose of this study was to compare the gene expression between TLR-2 and TLR-4 in amniochorion membranes of pregnant women with preterm delivery in the presence of histologic chorioamnionitis (HCA). Amniochorion membranes were collected from 40 pregnant women with preterm delivery;20 presented HCA and 20 did not. Fragments of the membranes were submitted to total RNA extraction, followed by cDNA production by reverse transcription. Real time quantitative PCR was used to quantify the gene expression of the TLRs. mRNA concentrations between TLR-2 and TLR-4 were compared using the nonparametric Mann-Whitney test. TLR-2 expression was higher than TLR-4 expression in the presence of HCA. No difference was observed between TLR-2 and TLR-4 expression in membranes in the absence of inflammatory infiltrate. In conclusion, amniochorion membranes express TLR-2 and TLR-4 and higher TLR-2 expression in the presence of histologic chorioamnionitis suggests that microorganisms recognizable by TLR-2 play an important role in the physiopathology of preterm labor.展开更多
文摘目的探讨脂多糖作用小鼠RAW264.7巨噬细胞24 h内细胞表面TLR4/CD14/MD-2受体复合物表达的变化特点。方法分别用低剂量(100 ng/ml)和高剂量(1 000 ng/ml)脂多糖刺激RAW264.7,应用RT-PCR检测TLR4、CD14、MD-2m RNA水平的变化,ELISA检测细胞上清TNF-α含量的变化。结果低剂量脂多糖下调TLR4 m RNA表达,14 h降至最低,24 h维持在低水平表达;CD14和MD-2 m RNA表达增加。高剂量脂多糖上调TLR4 m RNA表达,1 h上升至高峰,此后维持在高水平表达;CD14和MD-2 m RNA表达也明显增强。高剂量脂多糖作用后TNF-α含量显著高于低剂量脂多糖(P<0.05)。结论高剂量脂多糖逆转低剂量脂多糖下调的TLR4表达,提高TLR4/CD14/MD-2受体复合物的表达水平,放大下游信号转导通路的炎症效应。
基金Supported by the National Natural Science Foundation of China,No.30300337,30200278,30170919
文摘AIM: To investigate the expression of toll-like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells (KCs)and their role in ischemia-reperfusion (IR) injury of ratliver graft.METHODS: KCs were isolated at 0 (control group), 2, 12,24 h (IR group) following IR in rat liver graft, mRNA expressionof TLR4 and MD-2 was detected by RT-PCR analysis, proteinexpression of TLR4/MD-2 was detected by flow cytometric(FCM) analysis, and tumor necrosis factor-(~ (TNF-(~) levelin supernatant was measured by ELISA. Then isolated KCswere incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-(~ level was measured again.RESULTS: The mRNA and protein expression of TLR4/MD-2 and the level of TNF-(~ in IR group increased significantlyat 2 h following IR, and reached the maximum at 12 h, andslightly decreased at 24 h, but were still significantly higherthan those in the control group (P<O.01). The expression ofthese factors was markedly decreased after anti-TLR4antibody treatment as compared with the IR group (P<O.01).CONCLUSION: Lipopolysaccharide (LPS) following IR canup-regulate TLR4/MD-2 gene and protein expression inKCs, and synthesize cytokine TNF-(~. Anti TLR4 antibodycan inhibit the production of TNF-(~ induced by LPS. TLR4and its partner molecule MD-2 may play an important rolein Kupffer cell activation and IR injury.
基金São Paulo Research Foundation(Fapesp)grants numbers 2008/55418-1 and 2008/53382-0.
文摘Expression of Toll-like receptors at the maternal-fetal interface during normal and complicated pregnancies has aroused interest in the last few years. However, despite the importance of TLR-2 and TLR-4, which recognizes most microorganisms presenting in the amniotic cavity infections associated with prematurity, comparison of the expressions of these receptors is rare in the literature. Thus, the purpose of this study was to compare the gene expression between TLR-2 and TLR-4 in amniochorion membranes of pregnant women with preterm delivery in the presence of histologic chorioamnionitis (HCA). Amniochorion membranes were collected from 40 pregnant women with preterm delivery;20 presented HCA and 20 did not. Fragments of the membranes were submitted to total RNA extraction, followed by cDNA production by reverse transcription. Real time quantitative PCR was used to quantify the gene expression of the TLRs. mRNA concentrations between TLR-2 and TLR-4 were compared using the nonparametric Mann-Whitney test. TLR-2 expression was higher than TLR-4 expression in the presence of HCA. No difference was observed between TLR-2 and TLR-4 expression in membranes in the absence of inflammatory infiltrate. In conclusion, amniochorion membranes express TLR-2 and TLR-4 and higher TLR-2 expression in the presence of histologic chorioamnionitis suggests that microorganisms recognizable by TLR-2 play an important role in the physiopathology of preterm labor.
