目的：探讨新型过氧化物酶Peroxiredoxin-1（Prx-1）对大鼠肺成纤维细胞（FB）增殖和JNK通路的影响。方法体外培养FB，随机分为：对照组、TGF-β1组、空转染组、Prx-1转染组。对照组：以0.4％血清浓度MEM作为基础培养液；TGF-β1组：0.4％血清培养条件下，给予TGF-β1（5μg/L）孵育细胞；空转染组：利用脂质体Lipo2000将空载体转染到FB 36 h，给予同步化处理12 h后，在0.4％血清培养条件下，给予TGF-β1（5μg/L）孵育细胞；Prx-1转染组：Prx-1真核表达质粒转染到FB 36 h，给予同步化处理12 h后，在0.4％血清培养条件下，给予TGF-β1（5μg/L）孵育细胞。质粒转染采用脂质体转染法；免疫荧光检测质粒转染和8-OhdG（DNA氧化产物）的水平；MTT法检测FB增殖；Western blot 检测JNK和Prx-1的表达情况。结果质粒成功转染入FB，转染Prx-1真核表达质粒使Prx-1在FB内蛋白表达水平增高。与对照组比较，TGF-β1组的FB增殖、8-OhdG及磷酸化的JNK表达均明显增加。与TGF-β1组比较，空转染组中的上述观察指标无明显变化，但在Prx-1转染组，这些指标均明显下降。结论 TGF-β1能够诱导FB生成反应性氧物质（ROS），并由此促进JNK的激活和FB增殖，而Prx-1可通过抑制ROS来抑制TGF-β1诱导的FB增殖。%Objective To investigate the effects of Peroxiredoxin -1 (Prx-1) on proliferation and JNK pathway of pulmonary fibroblasts ( FB) of rats.Methods Cultured FB were randomly divided into four groups , control:0.4%fe-tal bovine serum;TGF-β1:final concentration is 5μg/L;TGF-β1 +vehicle:the empty plasmid was transected into FB for 36 h and then cells were treated with TGF -β1 (5 μg/L);TGF-β1 +Prx-1 plasmid:the plasmid including Prx-1 gene was transected into FB for 36 h and then cells were treated with TGF -β1(5μg/L).The plasmid was transfected in-to FB using the liposome infection .Immunofluorescence was used to evaluate plasmid transfection and 8-OhdG levels in FB;while MTT assay was used to evaluate FB proliferation and western blot was used to evaluate the expressions of phos -phorylated JNK ( p-JNK) , total JNK and Prx-1.Results The plasmids were successfully transfected into FB , showing as green florescence in cytoplasm and increased expression of Prx -1 protein.TGF-β1 significantly stimulated FB prolif-eration, and up-regulated the expression of p -JNK and 8 -OhdG in FB, which were markedly inhibited by Prx-1 plasmid transfection but not vehicle .Conclusion TGF -β1 induces FB to generate reactive oxygen species ( ROS ) , which contributes to JNK activation and FB proliferation;however , Prx-1 overexpression inhibits these changes by reduc-ing ROS level.
展开▼
