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The Study of Inhibiting Effect about NF-κB Decoy Oligonucleotides on the Fibroblast Cell’s Collagen Expression

Author: YangXueMei
Tutor: CuiSheHuai
School: Third Military Medical University
Course: Internal Medicine
Keywords: Trapping oligonucleotide NF-κB Lung fibroblasts Primary cultured Collagen type I Collagen type III Pulmonary fibrosis
CLC: R563.9
Type: Master's thesis
Year: 2008
Downloads: 71
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Abstract


Lung fibroblast collagen secretion increased, resulting in excessive deposition of extracellular matrix pulmonary fibrosis (Pulmonary Fibrosis, PF) pathomorphology its secretion of type I and type III collagen. The increase in lung fibroblast collagen secretion is a key link of pulmonary fibrosis, reduce lung fibroblast collagen expression is considered to be an effective way for the treatment of pulmonary fibrosis. Cytokine network plays an important and complex role in the occurrence and development of lung fibrosis, lung fibroblast collagen secretion in a variety of cytokines, transforming growth factor-β1 (Transforming Growth Factor-β1, TGF-β1) the impact of the biggest role. Therefore, the signal transduction pathways in pulmonary fibrosis research focus in recent years. Nuclear factor-κB (Nuclear Factor-κB, NF-κB) with a variety of cytokines upstream promoter-specific binding sites, thereby regulating the gene expression of multiple cytokines. NF-κB Upregulation of TGF-β1 gene expression in multiple experimental studies confirmed that in view of the important role of TGF-β in pulmonary fibrosis by inhibiting the activity of NF-κB inhibition of TGF-β signaling Upregulation of lung fibroblast collagen secretion, the way to reducing lung fibroblast collagen secretion, is expected to become effective treatment strategies for pulmonary fibrosis. The purpose of this study by cationic liposome-mediated NF-κB the trapping oligonucleotide Policy (decoy-oligonu--cleotides, Decoy ODNs) inhibited in vitro lung fibroblasts NF-κB activity observed lung fibroblasts collagen type I and type III collagen expression level changes, explore new therapeutic approach for the treatment of pulmonary fibrosis. The subject in the following two aspects: 1 block method of application of lung tissue, poor adherence method in primary culture, purified mouse lung fibroblasts ODNs-liposome complex, TGF-β5ng/ml stimulated was intervention by gel retardation (Electro-phoretic Mobility Shift Assay, EMSA) detection of NF-κB activity under the conditions of different doses and at different time points; 2 by RT-PCR reaction for detection of NF-κB activity after inhibition of lung fibroblast collagen Ⅰ and Ⅲ collagen mRNA expression. Main results: 1 application of lung tissue explant and poor adherence when combined primary cultured mouse lung fibroblasts obtained high purity lung fibroblasts. Confirmed by EMSA detection of NF-κB activity, compared with the control group, the decoy ODN-liposome complexes intervention after lung fibroblasts, NF-κB activity decreased significantly (P lt; 0.05). decoy ODN concentration 2μg/ml, NF-κB activity decline does not reach statistical significance (P gt; 0.05), the concentration of NF-κB 4μg/ml when activity decreased (P lt; 0.05), but the concentration of 8μg/ml NF-κB activity minimum. can be observed to the activity of NF-κB decoy ODN intervention After 2 hours decreased after 24 hours of NF-κB activity has not been restored. 3 by reverse transcription-polymerase chain reaction (reverse transcriptase-polymerase chain reaction, RT-PCR) reaction for detection of NF-κB activity was inhibited lung fibroblast collagen Ⅰ and Ⅲ collagen mRNA expression levels of NF-κB activity was inhibited collagen type I and type III collagen mRNA expression were decreased (P lt; 0.05), the expression levels of NF-κB activity was positively correlated. The decoy ODN intervention after two hours, the amount of collagen type I and type III collagen mRNA expression decreased obvious, six hours after the intervention, the amount of collagen mRNA began to decline, to 24 hours after the intervention, the amount of collagen mRNA expression is still at a low level. The main conclusions are: 1. Lung tissue explant adherent difference when combined applications can be effectively separated, purified mouse lung fibroblast cells. 3 to 5 cells suitable for experimental lung fibroblast cells were observed. Cationic liposome-mediated NF-κB the trapping oligonucleotide strategy can effectively inhibit the activity of NF-κB. Relative to the decoy ODN concentration of 2μg/ml and 4μg/ml, 8μg/ml the inhibition of NF-κB activity. 3.decoy ODN role in early lung fibroblasts that play the inhibition of NF-κB, adding decoy ODN 2 hours after the lowest activity of NF-κB, NF-κB inhibition lasts at least 22 hours. decoy ODN to provide basic data for further application of the method of the NF-κB activity relationship of aging. Inhibition of NF-κB activity can effectively reduce the in vitro lung fibroblast collagen Ⅰ and Ⅲ collagen mRNA expression, suggesting that inhibition of NF-κB activity can reduce the deposition of extracellular matrix in the process of pulmonary fibrosis. 5.decoy ODN role in lung fibroblasts 6 hours can inhibit collagen mRNA expression, inhibition lasts at least 18 hours or so. Collagen mRNA expression levels of NF-κB activity was positively correlated with further evidence of inhibition of NF-κB activation strategy to delay the development of pulmonary fibrosis, is expected to become one of the effective treatment of pulmonary fibrosis. Value needs further study to determine its practical application.

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