| Objective:(1) Meta-analysis of the relationship by ADAM33gene T1(rs2280091), S1(rs3918396) and S2(rs528557) loci associated with chronic obstructive pulmonary disease susceptibility.(2) in patients with chronic obstructive pulmonary disease Uygur and Uygur normal primary lung fibroblasts were cultured in vitro identification.(3) through the expression of Uighur patients with chronic obstructive pulmonary disease and normal lung fibroblasts Uygur ADAM33protein and gene explore whether Uygur patients with chronic obstructive pulmonary disease in primary lung fibroblasts in the presence of high expression of ADAM33.(4) investigate whether Thl/Th2cytokine expression regulation by ADAM33’s role in lung fibroblasts, thus affecting airway remodeling, thereby inhibiting or promoting the development of COPD, for further study ADAM33and T cell factor on airway remodeling mechanism of action, and to help develop treatments for chronic obstructive pulmonary disease provide a theoretical basis for clinical practice.Methods:(1)Correlation between Pubmed database, Embase databases, China National Knowledge Infrastructure Database (CNKI) and WanFangDATA published before September5,2012relating to ADAM33gene polymorphisms with chronic obstructive pulmonary disease susceptibility literature, two independent staff carefully evaluate all of the studies, a clear composite inclusion criteria, extracted the following information for each study:first author’s name, year of publication, race, number of cases and controls, chronic obstructive pulmonary disease genotype definition, case and control groups using METAGEN (STATA12.0) and Revman5.0software for statistical analysis.(2) by the First Affiliated Hospital of Xinjiang Medical University, Department of Thoracic Surgery provide normal lung tissue due to lung cancer resection specimens. Separation of adherent patients with chronic obstructive pulmonary disease Uygur and Uygur normal lung fibroblasts cultured in vitro, using the confocal microscope were identified.(3) using RT-PCR, Western blot method for determining Uighur patients with chronic obstructive pulmonary disease and normal lung fibroblasts Uygur level of ADAM33.(4) was added at the same concentration of normal lung fibroblasts Uygur medium, cytokines (IL-4, IL-13, INF-γ)(100ng/ml), was added before the24hours of serum starvation cytokines stimulate different after time (Oh,6h,12h,48h) cells were collected, using RT-PCR, Western blot of lung fibroblasts level of ADAM33; adding different concentrations of cytokines in the normal lung fibroblasts Uighur culture medium (IL-4, IL-13, INF-γ)(0,1ng/ml,10ng/ml,100ng/ml), was added before the cytokine serum starved for24hours after the stimulation cells were collected24h, using RT-PCR, Western blot of lung fibroblasts level of AD AM33.Results:(1) through the literature contained in the10and3765in2139COPD control group, the results show that S2(rs528557) and T1(rs2280091) and did not increase or decrease the risk of chronic obstructive pulmonary disease. In terms of race stratification, S1(rs3918396)(GG+AG vs. AA) in Asian populations and the general population associated with susceptibility to COPD. ORtotal=1.27(95%CI1.03-1.56, P=0.03), ORAsian=1.44(95%CI1.13-1.83, P=0.003), but does not exist in the Caucasian population related.(2) vimentin immunofluorescence confocal imaging results suggest that lung tissue from primary cultured cells isolated more than99%of vimentin.(3) by fluorescence quantitative RT-PCR analysis of ADAM33gene in patients with primary pulmonary Uygur COPD fibroblasts and normal lung fibroblasts expression patterns, P=0.042<0.05, the difference was statistically significant. Meanwhile, the use of Western blot technique to detect ADAM33protein in patients with primary pulmonary Uygur COPD fibroblasts and Uighur normal lung fibroblasts expression, P=0.011<0.05, the difference was statistically significant.(4) With INF-y stimulation time increases (Oh,6h,12h,48h), the expression of ADAM33gene was significantly reduced, INF-y stimulation time with2-(△△Ct) was negatively correlated (r=-0.399, P=0.039); Similarly, as INF-y stimulation time increases (Oh,6h,12h,24h,48h), the expression of ADAM33protein was significantly reduced, INF-y stimulation time and protein expression was negatively correlated (r=-0.268,P=0.048). INF-y stimulation with increasing concentrations (Ong/ml,1ng/ml,10ng/ml,100ng/ml), expression of the ADAM33gene significantly decreased, INF-y stimulation concentration2-(△△Ct) was negatively correlated (r=-0.492, P=0.027); Similarly, stimulation with INF-y concentration increased (Ong/ml,1ng/ml,10ng/ml,100ng/ml), ADAM33protein expression was significantly reduced, INF-y stimulation time and protein expression was negatively correlated (r=-0.558, P=0.011). With IL-4stimulation time increases (Oh,6h,12h,48h), the expression of ADAM33gene significantly increased, IL-4stimulation time with2-(△△Ct)were positively correlated (r=0.888, P=0.041); Similarly, as IL-4stimulation time increases (Oh,6h,12h,24h,48h), the expression of ADAM33protein was significantly increased, IL-4stimulation time and protein expression was positively correlated (r=0.703, P=0.038). Stimulation with IL-4concentration is increased (Ong/ml,1ng/ml,10ng/ml,100ng/ml), expression of the ADAM33gene significantly increased, IL-4stimulation of concentration2-(△△Ct) was positively correlated (r=0.383, P=0.004); Similarly, stimulation with IL-4concentration is increased (Ong/ml,1ng/ml,10ng/ml,100ng/ml) expression, ADAM33protein volume increased significantly, IL-4stimulation time was positively correlated with protein expression (r=0.508,.P=0.003). IL-13stimulation time with2-(△△Ct) no correlation (r=-0.254,P=0.108); same, IL-13stimulation time no correlation with IOD values (r=-0.189, P=0.453). Stimulated with IL-13concentrations2-(△△Ct) no correlation (r=-0.535, P=0.37); same, IL-13stimulation time no correlation with IOD values (r=-0.351, P=0.08).Conclusion:(1) through the database associated with COPD and meta-analysis of studies of ADAM33, ADAM33and studied the association of chronic obstructive pulmonary disease susceptibility. Our findings indicate that, S2(rs528557), T1(rs2280091) and homozygous carriers did not increase or reduce the risk of chronic obstructive pulmonary disease. Analysis described in this report show that was significantly associated with chronic obstructive pulmonary disease in Asian populations S1(rs3918396)(GG+AG vs. AA).(2) isolation and culture of Uighur Uighur patients with COPD and normal primary lung fibroblasts.(3) ADAM33in Uygur patients with COPD in primary lung fibroblasts expression levels than normal lung fibroblasts Uygur expression significantly.(4) IFN-y may inhibit the expression of ADAM33, in lung fibroblasts; IL-4may be the contrary, by increasing the expression of ADAM33, in lung fibroblasts, and thus airway remodeling, thereby inhibiting or promote the development of COPD, and provide a theoretical basis for further study of the mechanism of ADAM33and T cell factor on airway remodeling, and to help develop treatments for chronic obstructive pulmonary disease in clinical practice. |
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