PEP-1-SOD1融合蛋白抑制血管紧张素Ⅱ诱导的大鼠心脏成纤维细胞Ⅰ型胶原的合成

摘要

目的 研究穿透肽(PEP-1)介导的铜-锌超氧化物歧化酶1(SOD1)(PEP-1-SOD1)融合蛋白对血管紧张素Ⅱ(ANGⅡ)诱导的大鼠心脏成纤维细胞(CFbs)Ⅰ型胶原合成的影响,并探讨其机制.方法 利用基因工程技术表达和纯化PEP-1-SOD1融合蛋白.提取原代大鼠CFbs并传代培养,采用2～5代细胞进行实验,实验分为对照组、ANGⅡ诱导组、SOD1预处理组和PEP-1-SOD1预处理组.SOD1预处理组和PEP-1-SOD1预处理组细胞分别以2 μmol·L-的SOD1和PEP-1-SOD1预处理2h后,再给1μmol·L-1的ANGⅡ诱导24h,用免疫荧光法检测细胞内超氧阴离子(O2-)水平,微量丙二醛(MDA)检测试剂盒检测各组细胞MDA含量.结果 Western blot和细胞免疫荧光结果显示PEP-1-SOD1成功穿透入CFbs; Western blot结果显示,与ANGⅡ诱导组比较,PEP-1-SOD1预处理组Ⅰ型胶原和α平滑肌肌动蛋白表达显著降低(P＜0.01),细胞内O2-和MDA水平也显著降低(P＜0.01).结论 PEP-1-SOD1融合蛋白穿透人大鼠心脏CFbs内,通过降低细胞内O2-水平,抑制CFbs的激活,减少Ⅰ型胶原的合成.%Objective To investigate the effect of PEP-1-SOD1 fusion protein on synthesis of collagen Ⅰ of rats induced by angiotensin Ⅱ (ANG Ⅱ) in rat cardiac fibroblasts(CFbs) and discuss the mechanism.Methods The genetic engineering technique was used to express and purify PEP-1-SOD1 fusion protein.Rat CFbs obtained from original generation of rats were cultured and passaged,and the cells from passage 2 to passage 5 were divided into four groups:control group,ANG Ⅱ induction group,SOD1 treatment group and PEP-1-SOD1 treatment group.The last two groups were pretreated with 2 μmol · L-1 SOD1 and PEP-1-SOD1 for 2 hours respectively,and induced by 1 μmol · L-1 angiotensin Ⅱ for 24 hours.And the levels of maleic dialdehyde (MDA) and O2-in cells were assayed with minimal MDA assay-kit and immunocytofluorescence,respectively.Results CFbs were penetrated successfully by PEP-1-SOD1.Compared with those in ANG Ⅱ induction group,the contents of collagen Ⅰ and alpha-smooth muscle actin reduced significantly (P ＜ 0.01),and the levels of MDA and O2-in cells also decreased significantly in PEP-1-SOD1 treatment group(P ＜ 0.01).Conclusion PEP-1-SOD1 fusion protein penetrated into rat CFbs suppresses activation of CFbs and lessens synthesis of collagen Ⅰ by decreasing the levels of O2-in cells.