摘要
目的寻找防治肺纤维化的新途径。方法体外培养肺成纤维细胞(PFB),转入核转录因子AP1顺式诱骗元件(AP1Decoy)后再加入博莱霉素(BLM)进行刺激,采用酶谱图法测定细胞培养上清液中基质金属蛋白酶2(MMP2)活力,ELISA法测定金属蛋白酶组织抑制剂1(TIMP1)蛋白量,RTPCR法检测MMP2和TIMP1的mRNA水平。结果BLM作用12h,PFB分泌的MMP2活力(平均吸光度值,0.77±0.08)比对照组(0.65±0.07)明显增强,差异有统计学意义(P<0.05);但转入野生型AP1Decoy后,MMP2的酶活力增强被抑制(为0.68±0.05),与对照组的差异无统计学意义(P>0.05)。BLM作用12h或24h,均使PFB分泌的TIMP1蛋白量[(39.3±4.3)、(46.3±4.8)ngml]及TIMP1mRNA的表达量(与βactin的条带积分吸光度比值,0.94±0.13、1.08±0.06)比对照组[(28.9±2.7)、(31.6±2.4)ngml和0.76±0.07、0.75±0.08]增高,差异均有统计学意义(P<0.05或P<0.01);转入野生型AP1Decoy后,TIMP1蛋白及mRNA的表达量与对照组接近;转入突变型AP1Decoy组,其MMP2的酶活力、TIMP1蛋白及mRNA的表达量均与单纯BLM刺激组接近。结论AP1Decoy对BLM引起的肺成纤维细胞MMP2酶活力升高及TIMP1的表达增强有抑制作用。
Objective To investigate the effect of AP-1 Decoy on matrix metalloproteinase 2（MMP-2） and tissue inhibitor of metalloproteinase （TIMP-1 ） imbalance induced by bleomycin-A5 （BLM-AS） in pulmonary fibroblasts. Methods Pulmonary fibroblasts were primary cultured,and transferred with AP-1 Decoy before treated with BLM-AS.MMPs activity in medium was determined by gelatin zymography. Protein content of TIMP-1 in medium was detected by ELISA.Expression of MMP-2 mRNA and TIMP-1 mRNA were determined by reverse transcriptase-polymerase chain reaction（RT-PCR）. Results BLM-A5 induced the increase in activity of MMP-2 at 12 h [ A： （0.77±0.08）vs （0.65± 0.07） P 〈 0.05] ,but it was suppressed by AP-1 Decoy[A： （0.68 ± 0.05）] .BLM-A5 up-regulated the expression of protein and mRNA of TIMP- 1 after 12 h, and 24 h [ （39.3 ± 4.3）, （46.3 ± 4.8） ng/ml vs（28.9 ± 2.7）, （31.6 ± 2.4） ng/ml] and [ Absorbance ratio to β - actin ： （0.94 ± 0.13,1.08 ± 0.06）vs（0.76 ± 0.07,0.75 ± 0.08）] （ P 〈 0.05 or P 〈 0.01 ）but AP-1 Decoy modulated the up-regulation. All these indexes in AP-1 Decoy group had no significant difference in contrast to the normal group. Mutant AP-1 Decoy had not the same function as AP-1 Decoy on the expression of MMP-2 and TIMP-1 in pulmonary fibroblasts. Conclusion AP-1 Decoy inhibits the increase in MMP-2 activity and the up-regulation of TIMP-1 induced by BLM-A5 in pulmonary fibroblasts.
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
北大核心
2005年第4期282-285,共4页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
卫生部呼吸系疾病重点实验室开放基金资助项目(WHK03016)
