Objective: To construct the multi probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD 2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods: The...Objective: To construct the multi probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD 2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods: The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD 2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1 3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD 2 mRNAs in both constitutive and inducible types.展开更多
Thermal injury is able to induce the immune functional inhibition in the host, but the mechanisms are largely unknown. The available evidence indicates that macrophages play an important role in immune regulations and...Thermal injury is able to induce the immune functional inhibition in the host, but the mechanisms are largely unknown. The available evidence indicates that macrophages play an important role in immune regulations and host defenses. Thus, studies of the effect of thermal injury on macrophages are essential. Transcriptional factors of AP-1 (activator protein-1) and NF-кB (nuclear factor-кB) are critical members of signal transduction pathways. These transcriptional factors have been shown to be necessary for a variety of expressions of cytokine genes. In this investigation, the effect of thermal injury on AP-1 and NF-кB was studied using a model for the murine thermal injury. Our findings indicated that thermal injury mediated the inhibition of AP-1 and NF-кB DNA binding activities and c-fos and IL-12 p40 gene expressions in response to LPS.展开更多
We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-κB activation induced by LPS and IFN-γ/...We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-κB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expression of IL-12 p40 and p35 mRNA, and the degradation of κBα induced by LPS or LPS/IFN-γ. EMSA showed that LPS could augment the NF-κB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-κB activity nor promote the degradation of kBa. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided with the high-level secretion of IL-12 p70 induced by IFN-γ/LPS; (ii) NF-KB signal pathway is essential for IFN-γ/LPS to induce IL-12 mRNA expression; (iii) by blocking the degradation of κB, the PSI suppresses the IL-12 p40/p35 mRNA expression induced by LPS and IFN-γ/LPS; (iv) NF-KB signal may not be involved in the mechanism by which IFN-γ enhanced the expression of the LPS-induced IL-12 p40/p35 mRNA.展开更多
文摘Objective: To construct the multi probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD 2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods: The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD 2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1 3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD 2 mRNAs in both constitutive and inducible types.
基金This work was supported by the Ministry of Science and Technology of China
文摘Thermal injury is able to induce the immune functional inhibition in the host, but the mechanisms are largely unknown. The available evidence indicates that macrophages play an important role in immune regulations and host defenses. Thus, studies of the effect of thermal injury on macrophages are essential. Transcriptional factors of AP-1 (activator protein-1) and NF-кB (nuclear factor-кB) are critical members of signal transduction pathways. These transcriptional factors have been shown to be necessary for a variety of expressions of cytokine genes. In this investigation, the effect of thermal injury on AP-1 and NF-кB was studied using a model for the murine thermal injury. Our findings indicated that thermal injury mediated the inhibition of AP-1 and NF-кB DNA binding activities and c-fos and IL-12 p40 gene expressions in response to LPS.
文摘We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-κB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expression of IL-12 p40 and p35 mRNA, and the degradation of κBα induced by LPS or LPS/IFN-γ. EMSA showed that LPS could augment the NF-κB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-κB activity nor promote the degradation of kBa. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided with the high-level secretion of IL-12 p70 induced by IFN-γ/LPS; (ii) NF-KB signal pathway is essential for IFN-γ/LPS to induce IL-12 mRNA expression; (iii) by blocking the degradation of κB, the PSI suppresses the IL-12 p40/p35 mRNA expression induced by LPS and IFN-γ/LPS; (iv) NF-KB signal may not be involved in the mechanism by which IFN-γ enhanced the expression of the LPS-induced IL-12 p40/p35 mRNA.
