Overexpression Of Mclca3 In Airway Epithelium Of Asthmatic Murine Models With Airway Inflammation

Objective: To study time-dependent expression of asthma-related gene mCLCA3 in airway epithelium of asthmatic murine models and to evaluate the correlation between mCLCA3 mRNA with several inflammatory factors.Methods: Thirty SPF BALB/ C mice were randomly divided into five groups (n = 6/group): D group (sensitized but unchallenged); E group(challenged for 1 days); F group (challenged for 3 days); G group (challenged for 5 days); H group (challenged for 7 days). The mRNA expression and protein location of mCLCA3 in the lung tissues were determined by RT-PCR and IHC respectively. Inflammatory cell infiltrates in lung tissues were observed by both H.E staining of lung tissue sections and flow cytometric detection of lung mononuclear cell suspensions, while differential cell counts in BALF were used to assess airway inflammatory cells. RT-PCR and intracellular staining were adopted to examine Th1and Th2 cytokines IL-4 and IFN-γ.The different types of chemokine gradients in inflammatory site were evaluated by ELISA.Results: The mRNA and protein expression of mCLCA3 were markedly induced from group E and the levels were time-dependent with challenge times; furthermore, mCLCA3 protein expressed exclusively in the airway epithelium. Significantly increased levels of inflammatory cell infiltrates in challenged groups (group E,F or G,H) compared with sensitized but unchallenged group (group D) were found both in lung tissue and in BALF, especially lymphocytes and eosinophils in group G,H as well as CD4+T cells in group F. The Th1 cytokines (IFN-γ) and Th2 cytokines (IL-4) in lung tissues increased in different degrees. There was no significant increase of IFN-γ, but of IL-4 in group H. The levels of CCL5,CCL17 and CCL22 were all higher in challenged group (especially CCL17 in group G,H, CCL22 in Group H, p<0.05) than in group D. mCLCA3 mRNA level was positively correlated with CCL17,CCL22 protein levels and total cell,eosinophil counts in all groups. Meanwhile, mCLCA3 mRNA levels were positively correlated with CCL5 in group E,F,G but negatively correlated with CD4+T in group F,G,H. There was no significant correlation between mCLCA3 and IL-4 or IFN-γ.Conclusion: Time-dependent overexpression of mCLCA3 in airway epithelium of asthmatic murine models may modulate the recruitment of inflammatory cells by regulating the function of chemokines derived from airway epithelial cells, which provides us some new clues for our further study on its effect in the pathogenesis of asthma. Objective: To construct recombinant adenovirus shuttle plasmid pDC315-SPA-mCLCA3 and study the target expression of mCLCA3 in airway epithelial cell.Methods:SPA-mCLCA3 gene was synthesized with reverse transcription polymerase chain reaction (RT-PCR) from PGL-SPA and pCR-BluntII-TOPO vectors, then was cloned into pDC315-EGFP to form pDC315-SPA-mCLCA3 (constructed by Genechem Co.). After identification with nucleotide sequencing analysis, the vector and control vector(pDC315-EGFP, acts as negative control as well as GFP positive control)were transfected into H441 and 293 cell lines with lipofectamine 2000 respectivly. mRNA levels of mCLCA3 in each cell line were determined by RT-PCR. Immunocytochemistry was applied to detect mCLCA3 protein in transfected cell line H441. Results:The recombinant plasmid was confirmed containing SPA promoter and mCLCA3 fusion gene. The expression of mCLCA3 mRNA was undetectable in two cell lines in control vector treated group, whereas in pDC315-SPA-mCLCA3 group, mCLCA3 mRNA increased significantly in H441 compared with that in 293 cell line (p<0.05), which confirmed its high transcriptional targeting activity in airway epithelial cell line. The result of immunohistochemical showed that mCLCA3 protein expression was not detected in control group while strong immunoreactivity was detected in pDC315-SPA-mCLCA3 group in H441 cell line.Conclusion: We have constructed recombinant adenovirus shuttle plasmid pDC315-SPA-mCLCA3 with airway epithelial cell-specific expression function, which may be helpful to the next study about the construction of the adenovirus SPA-mCLCA3 and the effect of mCLCA3 to abnormal airway epithelial cells in vivo.