低氧对心脏成纤维细胞增殖细胞核抗原表达与表型的影响

Effect of hypoxia on the expression of proliferating cell nuclear antigen and phenotype of cardiac fibroblasts

目的 :观察不同程度低氧对乳鼠心脏成纤维细胞 (CFs)增殖细胞核抗原 (PCNA)表达与表型的影响。方法 :分离、纯化、培养乳鼠CFs ,随机分为 3组 :对照组、中度低氧组和重度低氧组 ,以四唑盐 (MTT)比色法检测细胞的增殖情况 ,采用免疫组织化学的方法检测PCNA的表达情况 ,电镜观察细胞的超微结构 ,并采用激光共聚焦显微镜检测各组细胞内α -actin的表达情况。结果 :中度低氧组MTT的 4 90nm吸光度值 (A490nm)较对照组增加 (18 4±2 5 0 ) % (P <0 0 5 ) ,而重度低氧组A4 90nm值较对照组降低 (15 8± 2 5 0 ) % (P <0 0 5 )。免疫组化结果显示 :对照组CFs的PCNA有一定的基础表达 ,核内阳性反应颗粒较少 ,染色强度为 (92 2± 9 1) %AU ,中度低氧组CFs的PCNA染色强阳性 ,可见密集的阳性反应颗粒 ,染色强度为 (12 8 0± 11 4 ) %AU(P <0 0 5vs对照组 ) ,重度低氧组CFs核内无明显的阳性反应颗粒 ,染色强度为 (89 9± 8 4 ) %AU。透射电镜下 ,正常组CFs呈梭形 ,胞浆内有丰富的粗面内质网和高尔基复合体 ,线粒体和微丝的含量较少 ,无基膜存在 ,呈典型的成纤维细胞表型。中度低氧刺激 72h ,CFs粗面内质网明显减少 ,胞浆内出现大量的微丝 ,以质膜下分布最多 ,呈现肌成纤维细胞表型。重度低氧组CFs内仅有少量的?

AIM:To investigate effect of hypoxia on the expression of proliferating cell nuclear antigen(PCNA) a nd phenotype of cardiac fibroblasts(CFs). METHODS: The purif ied cardiac fibro blasts were cultured and divided randomly into there groups :control group, mode rate hypoxia(MH) group and severe hypoxia(SH) group. After 72 h,MTT method was u s ed to investigate the proliferation of CFs, and the ultrastructure of fibroblast s were observed with transmission electron microscopy The expression of PCNA a n d α-actin in cardiac fibroblasts were measured by the means of immunohistochemi s try and laser scanning confocal microscopy, respectively. RESULTS: MTT A 490 nm value of MH group was significantly higher than that of control group by (18 4±25 0)% ( P< 0 05), whereas MTT A 490 nm value of MH group was signific a ntly lower than that of control group by (15 8±25 0)% ( P< 0 05) The immunohistochemistry experim ent showed that there was basal PCNA expression in control CFs, and it was incre ased in MH CFs( P< 0 05 vs control group), but there was no significant difference between SH and control CFs. Observed with transmission electron microscopy, the control CFs had typical phenotype of fibroblasts: shuttle-shaped, abundant roug h endoplasmic reticulums (RER) and golgi complexes,but few mitochondria or m icromyofilaments in cytoplasm. After MH for 72 h, there appeared lots of micromy o filaments in cytoplasm, but there were little micromyofilaments in SH CFs. Obser ved with laser confocal scanning microscopy, there was lower expression of α-ac tin in CFs of control group, while many regular bundles of micromyofilaments and the expression of α-actin were signifiantly increased ( P< 0.05 vs con trol group) in CFs treated with MH. The α-actin expression in severe hypoxia CFs was not sig nificantly different from control group. CONCLUSION: MH made C Fs have the ch aracteristics of myofibroblasts phenotype and enhanced PCNA expression.