| Fibroproliferative matrix molecule deposition and enhanced collagen accumulation of pulmonary fibrosis cells are the main pathomorphology character of pulmonary fibrosis, which mainly secrete type I and type III collagen. The enhanced collagen is the key point in thecourse of pulmonary fibrosis. So reducing the collagen was known as an effective way to treat pulmonary fibrosis. Much evidence proved that cytokine network plays an important role in the course of pulmonary fibrosis. Among all of the cytokines, transforming growth factor-β1(TGF-β1) has the most important effect on the pulmonary fibrosis. As a consequency, many researches focused on the cytokine in recent years.Nuclear Factor-κB (NF-κB) can combine with specific biding sides of many cytokine promoters, to adjust the cytokine gene expression. A great deal of evidences have proved NF-κB could adjust the expression of TGF-β1 gene. So according to the theory that TGF-β1 has the effect to the pulmonary fibrosis, the way to reduce the activity of NF-κB that reduce the enhanced collagen accumulation of pulmonary fibrosis,might be the effective strategy to treat pulmonary fibrosis.The aim of this work is to investigate the effect of NF-κB decoy -oligonucleotides (Decoy ODNs) on the activity of NF-κB and expression of fibroblast type I and type III collagen, and to provide the therapeutical foundation of lung fibrosis.This work completed two works: (1) Primary murine lung fibroblasts were purified by lung tissue method and different timely adhensive method, fibroblasts were transfected by ODNs-cationic liposomes complex after the stimilation of 5ng/ml TGF-β, and the inhibiting effect of Decoy ODNs on NF-κB was determined by electrophoretic mobility shift assay ( EMSA). (2) The effect of Decoy ODNs on the type I and type III collagen synthesis in the lung fibroblasts were analyzed by RT-PCR. The main results:(1) Primary murine lung fibroblasts were purified with high purity by lung tissue method and different timely adhensive method.(2) NF-κB Decoy ODNs could competitively inhibit the NF-κB activity compared with controls(P<0.05). The activity of NF-κB started to reduce when the concentration of Decoy ODN reached 2μg/ml(P>0.05), activity reduced when the concentration of Decoy ODN reached 4μg/ml(P<0.05),and the activity reached the min value when the concentration of Decoy ODN reached 8μg/ml(P<0.05). The activity of NF-κB started to reduce after inhibiting for 2 hrs, and the activity did not recovery after 24 hrs.(3) The RT-PCR assay showed the mRNA expressions of fibroblast type I and type III collagens decreased, the value have the positive correlation with the activity of NF-κB. The mRNA expression of fibroblast type I and type III collagen did not reduce obviously after inhibiting for 2 hrs,the mRNA expression started to reduce after inhibiting for 6 hrs, the mRNA expression remained low level after inhibiting for 24 hrs, the mRNA expression began to increase after inhibiting for 36 hrs.The main conclusion:(1) Lung tissue method combined with different timely adhensive method could be used to separate and purify murine lung fibroblasts. The 3th to 5th generation cells could be used as a model to observe.(2) NF-κB Decoy ODNs mediated by liposome could competitively inhibit the NF-κB activity. The 8μg/ml Decoy ODN had the optimal result compared with 2μg/ml and 4μg/ml Decoy ODN.(3) Decoy ODN could competitively inhibit the NF-κB activity at the early stage, the activity reached the minimum value after inhibiting for 6 hrs, and the inhibition effect lasted for 22 hrs. Compeititive inhibiting effect of Decoy-ODNs on the activity of NF-κB provided fundamental data for further application.(4) NF-κB Decoy ODNs could also competitively inhibit the mRNA expression of fibroblast type I and type III collagen, which might provide the therapeutic foundation of lung fibrosis.(5) NF-κB Decoy ODNs could competitively inhibit the mRNA expression of fibroblast I collagen after inhibiting for 6 hrs, and the inhibition effect lasted for 30 hrs which showed the inhibitition effect was time limited. The clinical worth need to be defined.
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