Toll signaling pathway may play a curcial role in induction of inflammation-associated gene activation. Originally, the Toll/spaetzle/Cactus-Dorsal signaling pathway is established in the Drosophila embryonic developm...Toll signaling pathway may play a curcial role in induction of inflammation-associated gene activation. Originally, the Toll/spaetzle/Cactus-Dorsal signaling pathway is established in the Drosophila embryonic development. Recently, the Toll signaling pathway in adult Drosophila has been established in the induction of antimicrobial peptide expression. Five human Toll-like receptor genes (h Tlr l-5 ) and one mouse Toll-like receptor gene (m Tlr-4 ) have been isolated. Toll and Toll-like receptor genes encoded molecules are transmembrane proteins with an extracellular leucine repeat domain and a cytoplasmic domain homologous to IL-1 receptors. The intracellular signaling cascade involves Tube, Pelle, and Cactus-Dorsal complex in Drosophila, and MyD88, IRAK, TRAF 6, NIK, αβ-I κB kinase, and I κB -NFκB complex in mammals. Dorsal and NFκB are transcription factors, while Cactus and IκB are their inhibitors. When the inhibitors phosphorylated, the nuclear factors are released and move into nucleus, leading to immune gene activation. It has been shown from in vitro system that Tlr -4 mediated LPS signaling in human monocytes for expression of IL-1, IL-6, IL-8, and costimulator B7-1 which provides second signal for T cell response. Tlr -2 can also mediate LPS signaling in human monocytes, leading to the production of proinflammatory mediators. Microbial lipoproteins are potent stimulators of IL-12 production through Tlr -2 signaling by human macrophages, and can stimulate Tlr 2-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Findings of a point mutation of Tlr-4 in LPS tolerant C3H/HeJ mouse strain and a deletion of Tlr-4 in LPS resistant C57BL/10ScCr mice provide an in vivo evidence strongly supporting the crucial role of Tlrs in LPS mediated inflammation. It is proposed that targeting Tlrs would develop new remedies for treatment of inflammatory disorders and for immunotherapy of mucosal infections and cancer, etc.展开更多
为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV....为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV.tag2 B构建出 h BD- 2的重组表达载体 p CMV.tag2 B/h BD- 2 ,并经 DNA测序证明h BD- 2 c DNA片段的插入方向和其全长 c DNA的碱基组成顺序均准确无误。通过脂质体转染法将 p CMV.tag2 B/h BD- 2导入 COS- 7细胞。RT- PCR法和免疫印迹法分别从 m RNA水平和蛋白质水平检测到被转染的 COS- 7细胞系能有效表达 h BD- 2。展开更多
β-防御素体外试验显示具广谱抗菌活性 ,本研究应用转基因方法评估其体内抗菌作用。构建大鼠 β-防御素 - 2 (r BD2 )重组质粒 p BK- CMV- r BD2和 p CDNA- 3.1- Myc- His(+) - r BD2 ,通过脂质体包裹的方法将重组质粒经气管滴入 ,检测...β-防御素体外试验显示具广谱抗菌活性 ,本研究应用转基因方法评估其体内抗菌作用。构建大鼠 β-防御素 - 2 (r BD2 )重组质粒 p BK- CMV- r BD2和 p CDNA- 3.1- Myc- His(+) - r BD2 ,通过脂质体包裹的方法将重组质粒经气管滴入 ,检测其在气管和肺组织表达情况 ,并应用肺组织匀浆上清菌落计数法检察对绿脓杆菌的肺清除率。结果显示在基因转染大鼠的气管和肺组织内均检测到 r BD2 - His融合蛋白基因 m RNA和蛋白的表达 ,说明脂质体包裹的重组 β-防御素 - 2 p BK- CMV- r BD2质粒可有效转染气道上皮组织。肺细菌清除率实验显示构建重组质粒 p BK-CMV- r BD2气道转染与对照相比可显著提高肺组织对绿脓杆菌的清除率 (n(8,p(0 .0 1)。本研究表明 β-防御素基因气道转染可提高呼吸道天然抗感染防御功能 ,可能在呼吸道感染的防治实践中具有潜在应用价值。展开更多
文摘Toll signaling pathway may play a curcial role in induction of inflammation-associated gene activation. Originally, the Toll/spaetzle/Cactus-Dorsal signaling pathway is established in the Drosophila embryonic development. Recently, the Toll signaling pathway in adult Drosophila has been established in the induction of antimicrobial peptide expression. Five human Toll-like receptor genes (h Tlr l-5 ) and one mouse Toll-like receptor gene (m Tlr-4 ) have been isolated. Toll and Toll-like receptor genes encoded molecules are transmembrane proteins with an extracellular leucine repeat domain and a cytoplasmic domain homologous to IL-1 receptors. The intracellular signaling cascade involves Tube, Pelle, and Cactus-Dorsal complex in Drosophila, and MyD88, IRAK, TRAF 6, NIK, αβ-I κB kinase, and I κB -NFκB complex in mammals. Dorsal and NFκB are transcription factors, while Cactus and IκB are their inhibitors. When the inhibitors phosphorylated, the nuclear factors are released and move into nucleus, leading to immune gene activation. It has been shown from in vitro system that Tlr -4 mediated LPS signaling in human monocytes for expression of IL-1, IL-6, IL-8, and costimulator B7-1 which provides second signal for T cell response. Tlr -2 can also mediate LPS signaling in human monocytes, leading to the production of proinflammatory mediators. Microbial lipoproteins are potent stimulators of IL-12 production through Tlr -2 signaling by human macrophages, and can stimulate Tlr 2-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Findings of a point mutation of Tlr-4 in LPS tolerant C3H/HeJ mouse strain and a deletion of Tlr-4 in LPS resistant C57BL/10ScCr mice provide an in vivo evidence strongly supporting the crucial role of Tlrs in LPS mediated inflammation. It is proposed that targeting Tlrs would develop new remedies for treatment of inflammatory disorders and for immunotherapy of mucosal infections and cancer, etc.
文摘为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV.tag2 B构建出 h BD- 2的重组表达载体 p CMV.tag2 B/h BD- 2 ,并经 DNA测序证明h BD- 2 c DNA片段的插入方向和其全长 c DNA的碱基组成顺序均准确无误。通过脂质体转染法将 p CMV.tag2 B/h BD- 2导入 COS- 7细胞。RT- PCR法和免疫印迹法分别从 m RNA水平和蛋白质水平检测到被转染的 COS- 7细胞系能有效表达 h BD- 2。
文摘β-防御素体外试验显示具广谱抗菌活性 ,本研究应用转基因方法评估其体内抗菌作用。构建大鼠 β-防御素 - 2 (r BD2 )重组质粒 p BK- CMV- r BD2和 p CDNA- 3.1- Myc- His(+) - r BD2 ,通过脂质体包裹的方法将重组质粒经气管滴入 ,检测其在气管和肺组织表达情况 ,并应用肺组织匀浆上清菌落计数法检察对绿脓杆菌的肺清除率。结果显示在基因转染大鼠的气管和肺组织内均检测到 r BD2 - His融合蛋白基因 m RNA和蛋白的表达 ,说明脂质体包裹的重组 β-防御素 - 2 p BK- CMV- r BD2质粒可有效转染气道上皮组织。肺细菌清除率实验显示构建重组质粒 p BK-CMV- r BD2气道转染与对照相比可显著提高肺组织对绿脓杆菌的清除率 (n(8,p(0 .0 1)。本研究表明 β-防御素基因气道转染可提高呼吸道天然抗感染防御功能 ,可能在呼吸道感染的防治实践中具有潜在应用价值。
