| Asthma is a kind of airway allergic inflammation disease associated with several inflammatory cells and structure cells. Among these cells, mast cells play an important role by releasing inflammatory mediators,cytokines and chemokines , resulting in airway obstruction,airway hyperresponsiveness and inflammation,thus initiating airway early phase response and participating in late phase response. Mast cells activation is mediated by Ag specific IgE and cytokines. Stem cell factor(SCF) is a main cytokine that activiates mast cells.Therefore, SCF may take part in pathophysiology process of asthma. In this experiment, in order to reveal the role of SCF in pathophysiology process of asthma and provide new target and method for asthma treatment , we observed the changes of SCF mRNA by semi-quantitive RT-PCR in OVA sentisized mice and dexamethasone-treated OVA sentisized mice, investigated the role of SCF-mediated interaction between fibroblasts and mast cells on asthma by antisene technology in vitro, and then, treated OVA sentisized mice with SCF ASON by intranasal administration.Methods and ResultsThe expression of SCF mRNA in OVA sentisized mice lung and effect of dexamethasone treatment on SCF mRNA By establishing OVA induced mice asthma by intranasal administration,we observed the effect of dexamethasone(2mg/kg,ip) on lung tissue pathology and determined the expression of mSCF mRNA and sSCF mRNA in lung tissue.As a result, 24 hours later, an infiltration of inflammatory cells, including eosinophils, lymphocte, neutrophil around bronchial, vessel and interstitium tissue of lung was observed in untreated OVA-sensitized mice. In contrast, inflammatory cells infiltration were lessened in lung tissue of dexamethasone treated mice.Compared with control mice ,mSCF mRNA increased(0.36±0.02 vs 0.16±0.03,P<0.01) and sSCF mRNA decreased(0.23±0.02 vs 0.41±0.05,P<0.01) in OVA-sensitized mice.After dexamethasone intervention,mSCF mRNA level dropped to near that of contol (0.18 ±0.03 vs 0.16±0.03,P>0.05),and sSCF mRNA increasd (0.87±0.04 vs 0.23±0.02,P<0.01). In vitro study for SCF mediated fibroblast-mast cell interaction We synthesized a 18-mer phosphorothioate antisense oligonucleotide(ASON) of SCF mRNA ,and then concubated NIH3T3 fibroblasts with cationic liposome(lipofectin)/ oligonucleotide complexes. The uptake of oligonucleotides in cells was determined by observing FAM-positive cells under fluoro-microscopy, SCF expression was determined by immunochemistry and RT-PCR respectively.Lipofectin(10ug/ml)/ SCF ASON(10ug/ml) complexes transfected fibroblast(NIH3T3 cells)-mast cell(mice bone marrow derived)cocultures, the growth of fibroblasts and mast cells was observed and growth curves were drawn.We determined mast cells apoptosis by electron microscopy, AO stain,flow cytometric analysis.Both histamine and eotaxin levels in culture supernatants of all groups were determined by ELISA and fluorometry.As a result, after SCF ASON/Lipofectin complexs transfection, FAM-labeled ASON distributed in the cytoplasm and to a lesser extent in the nucleus of cells with striking down-regulation of SCF protein and mRNA.However,these effects were not observed in lipfectin transfection alone.These data indicate that cationic liposome(lipofectin) increases the uptake of ASON and activity.Cultured NIH3T3 grew at one-layer pattern,NIH3T3 of fibroblast-mast cell coculture grew at multi-layers pattern.Lipofectin/SCFASON complexes inhibited growth of both NIH3T3 and mast cell of cocultures,and also induced mast cell apoptosis(14.0±0.81% at 96 hour point).Both histamine(3.83±0.41ng/ml vs 0.33±0.26ng/ml,P<0.01) and eotaxin (5.785±0.395ng/ml vs 1.875± 0.252ng/ml, P<0.01 )levels of fibroblast-mast cell cocultures supernatants,were much higher than that of fibroblast or mast cell cultures supernatants；and both histamine (3.08±0.38ng/ml,P<0.05)；and eotaxin ( 4.398±0.333,P<0.05 )levels of Lipofectin/SCFASON complexes treated fibroblast-mast cell cocultures s...
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