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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 find a new way to prevent and cure pulmonary fibrosis. Methods Pulmonary fibroblasts (PFB) were cultured in vitro and transfected with ap1 decoy (AP1Decoy). The cells were stimulated by BLM, and the concentration of stromal cells The activity of MMP2, the protein level of TIMP1 by ELISA and the mRNA level of MMP2 and TIMP1 by RTPCR. Results After treated with BLM for 12 hours, the activity of MMP2 secreted by PFB (mean absorbance value 0.77 ± 0.08) was significantly higher than that of the control group (0.65 ± 0.07) (P <0.05). However, after transfected with wild-type AP1Decoy, MMP2 (0.68 ± 0.05). There was no significant difference with the control group (P> 0.05). The BLM-induced TIMP1 protein levels [(39.3 ± 4.3), (46.3 ± 4.8) ng ml] and TIMP1 mRNA (the ratio of the absorbance of the bands to β-actin were 0.94 ± 0.13, 1.08 ± 0.06 ) Was significantly higher than that of the control group [(28.9 ± 2.7), (31.6 ± 2.4) ngml and 0.76 ± 0.07,0.75 ± 0.08, respectively, P <0.05 or P <0.01) , The expression of TIMP1 protein and mRNA were similar to that of the control group. The activity of MMP2 and the expression of TIMP1 protein and mRNA in the mutant AP1Decoy group were similar to those in the BLM-stimulated group. Conclusion AP1Decoy can inhibit the increase of MMP2 activity and the expression of TIMP1 induced by BLM in lung fibroblasts.