| Objective : Asthma is a complex inflammatory disease, which is increasing in freqnency and sevevity despite the significant advances in the understanding of its physiology and treatmant. Inhaled corticosteroids have dramatically improved the treatmant of asthma and now is the first-line drug for persistant asthma, for steroid-dependent asthmatics, high doses of oral corticosteroids are required , however, systematic side effects limit the doses that can be given for a long time, and corticosteroids has little effects on inflammation of steroid-resistant asthma. In order to better understanding the mechanisms of dexamethasone, try to find drugs with steroids-sparing and superceding effects, and to find the possibility of whether the adenovirus mediated recombinant sIL-4R gene could be used as a alternative therapy for asthma. Methods:sIL-4R eDNA fragment is synthesized from spleen cells of BALB/c murine stimulated with IL-4 in vitro using RT-PCR, the sequence of recombinant sIL-4R is indentified by sequence analysis . The cDNA fragment is inserted into the Hind III and Kpn I cloning sites of adenoviral shuttle vecotor pAdCMV Link]. A lacZ gene fragment from plasmid pcDNA3.1 vs/His LacZ is subcloned into Bgl II and ClaI cloning sites of pAdCMV Linkl vector. The plasmids are respectively cotransfected with adenoviral bone vector pJM 17 into 293 package cells mediated by Lipofectamine Plus regeant. Recombinant replication-deficant adenovirus are confinned by PCR or X-gal stain. Mouse model of asthma was copied according to the protocal of administration OVA as an allergen.Results:1. The sequence of recombinant sIL-4R shares 98% homology compared with the published data of secreted IL-4R from T cell line CTLL and share 100% homology with the extracellular part of IL-4R allotype, 9- base substitutions lead to 3- amino acid varies in the sIL-4R protein. 2. Adenoviral shuttle vectors containing murine sIL-4R and-5-LacZ gene are successfully constructed by restriction analysis. 3. 293 cells show cytopathic effect, recombinant replication-deficient adenovirus are obtained from the supernatant, or 293 cells become blue when stained with X-gal. 4. Murine asthma model is copied successfully, the inflammation in lung are obvious that inflammatory cells such as mast cells, eosinophiles and lymphocytes are infiltrated in the lung , especially in the areas around the blood vessels and bronchus detected by microscopy. 5. There is no difference in the expression level of IL-4Ra mRNA between the positive and negative control groups of asthma. Dexamethasone treatment before OVA challenge in OVA-immunized mice do not modify the IL-4Ra mRNA expression and the mRNA half-life, there are little differences in the IL-4Ra PCR product signals which are quantified by scanning densitometry and normalized as a percentage of the 13-actin PCR signal between positive control mouse and dexarnethasone treated mice. 6. The level of sIL-4R mRNA in OVA challenged mice is lower than negative mice at the 12th piont. Dexamethasone can upregulate the expression of sIL-4R mRNA in a time-dependent manner. The scanning signal of sIL-4R rnRNA PCR products at 12th hour in positive control groups is twenty-fold lower than that in dexamethasone treated group. Conclusions: We have made the basic foundation of gene therapy using sIL-4R gene for asthma. The results indicate that the dexamethasone inbibits IL-4R expression at posttranscriptional level and sIL-4R is a downstream effector of dexamethasone, exogenous sIL-4R protein may be considered as application for severious asthma.
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