氧化还原因子1促进乳鼠心肌成纤维细胞增殖  

Redox factor 1 increases proliferation of neonatal rat cardiac fibroblasts

作　　者：耿燕[1] 李涛[2] 胡晓青[1] 张晨光[1] 陈苹[1] 马康涛[1] 周春燕[1] 

机构地区：[1]北京大学基础医学院生物化学与分子生物学系,北京100191 [2]浙江师范大学化学与生命科学学院生物系

出　　处：《北京大学学报（医学版）》2009年第3期335-342,共8页Journal of Peking University:Health Sciences

基　　金：北京大学985研究经费资助~~

摘　　要：目的:研究氧化还原因子1(redox factor 1,REF1)对乳鼠心肌成纤维细胞增殖和胶原合成的影响及其可能的机制。方法:为证实REF1在乳鼠心肌成纤维细胞增殖和胶原合成中的作用,构建重组Ref1野生型和突变型的腺病毒载体,感染乳鼠心肌成纤维细胞。通过RT-PCR和Western印迹检测Ref1以及与胶原合成相关的基因表达的改变,通过免疫荧光检测REF1核转位,利用MTT和流式细胞仪分析细胞增殖和细胞周期,用电泳迁移率实验(electrophoretic mobility shift assay,EMSA)检测REF1对转录因子活化蛋白1(activator protein 1,AP1)的DNA结合能力的影响。利用含25 mmol/L的葡萄糖培养基模拟高糖环境,观察对乳鼠心肌成纤维细胞增殖及REF1表达与活性的影响。结果:MTT比色法显示,感染野生型Ref1重组腺病毒的细胞在490 nm处的光密度值明显高于对照病毒组(0.671±0.044vs0.364±0.007,n=6,P<0.01)。流式细胞仪对细胞周期的分析表明,野生型Ref1能明显增加S期细胞的百分率(16.8%±0.62%vs9.04%±0.43%,n=3,P<0.05)。RT-PCR检测发现,野生型Ref1促进细胞Ⅰ型胶原(collagen I,ColⅠ)和Ⅲ型胶原(collagenⅢ,ColⅢ)mRNA表达增多,但突变型Ref1对细胞的增殖和胶原的合成均没有明显影响。EMSA实验结果显示,感染野生型Ref1重组腺病毒的细胞AP1的DNA结合能力增强。高糖培养基培养乳鼠心肌成纤维细胞同样能增强AP1的DNA结合能力。高糖培养虽然对Ref1的表达水平没有影响,但增强了细胞中REF1的核转位,促进了细胞的增殖。结论:REF1促进乳鼠心肌成纤维细胞的增殖和胶原合成,其机制可能是通过上调AP1的DNA结合能力。Objective：To investigate the function of REF1 in the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts, and the underlying laechanisms. Methods： Neonatal rat cardiac fibro- blasts were transfected with the adenoviral vector containing rat wild type Ref1 （ Ad-Ref1 ） or mutated Refl （Ad-mutRefl）. The mutations resulted in Cys to Ala at amino acids 65 and 93, which eliminated the redox function of the REFI protein. MTF was used to check the cell viability and flow cytometry was used to analyze the cell proliferation with the count of cell numbers and the percentage of cells in S phase of the cell cycle. The expressions of Refl, collagen Ⅰ （Col Ⅰ ） and collagen Ⅲ （Col Ⅲ） were deter- mined by RT-PCR and Western blot. The translocation of REF1 was examined by fluorescence staining and revealed under fluorescence microscope. Electrophoretic mobility shift assay （EMSA） was used to check the effect of REF1 on AP1 DNA binding ability. The high glucose medium （25 mmol/L） was ap- plied to culture cardiac fibroblasts. The effect of high glucose on AP1 DNA binding activity, the expres- sion and translocation of REFl were examined. Results： MTT analysis showed that Ad-Refl promoted the relative viability of cardiac fibroblasts （0. 671 ± 0. 044 vs control 0. 364 ± 0. 007, n = 6, P 〈 0. 01 ）. The percentage of cells in S phase of the cell cycle was increased significantly in the Ad-Refl transfected cells （ 16.8% ±0. 62% vs control 9.04% ±0.43%, n =3, P 〈0. 05）, as demonstrated by flow eytometry a- nalysis. The expressions of Col Ⅰ and Col Ⅲ at mRNA level were increased when cells transfeeted with Ad-Refl, while Ad-mutRefl did not show such effects. Compared with the redox-defieient mutant Ad- mutRefl （C65/93A） , EMSA results demonstrated that Ad-Refl resulted in a marked increase in AP1 DNA binding. We also found that the cardiac fibroblasts cultured in high glucose （25 mmol/L） medium resulted in an increase in AP1 DNA binding activity, which

关 键 词：氧化还原因子1 成纤维细胞 细胞增殖 胶原 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]