肿瘤坏死因子α对新生大鼠心肌成纤维细胞增殖的影响及其机制

Effect of TNF-α on proliferation of rat cardiac fibroblasts and its underlying mechanism

目的:探讨肿瘤坏死因子α(TNF-α)刺激对新生大鼠心肌成纤维细胞(CFs)增殖的影响及其机制。方法:采用消化法培养新生SD大鼠的CFs。实验分为4组,即5、10和20μg/L TNF-α处理组及空白对照组。将CFs培养36 h后,用四氮唑蓝(MTT)比色法测定细胞的增殖;分别用半定量RT-PCR和Western blot技术检测在5μg/L和10μg/L TNF-α刺激下细胞周期蛋白D1(CyclinD1 mRNA及其蛋白)表达的变化。结果:随着TNF-α浓度的增高,MTT比色法检测的A490值呈明显递增趋势,其中5、10及20μg/L组的A490值,分别为0.417±0.011、0.622±0.015和0.602±0.013,与对照组(0.235±0.013)相比,有显著差异(P<0.05)。RT-PCR和免疫印迹检测表明,以5μg/L和10μg/L TNF-α刺激36 h后,CyclinD1 mRNA(0.706±0.113,1.698±0.135)和其蛋白(1.270±0.168,2.749±0.170)的水平均明显高于对照组CyclinD1mRNA(0.192±0.039)和蛋白(0.658±0.101)的表达水平(均P<0.01)。结论:TNF-α可通过上调CyclinD1的表达促进心肌成纤维细胞增殖,这可能为应用TNF-α信号通路抑制剂进行抗心肌纤维化治疗提供实验依据。

AIM： To investigate the effects of tumor necrosis factor α（TNF-α） on the proliferation of cardiac fibroblast （CF） and the underlying mechanism. METHODS： CFs of neonatal Sprague Dawley rats were isolated by enzymatic dissociation. CF proliferation was measured by thiazolyl blue （MTF） assay and expression of cyclinD1 was detected by RT-PCR and Western blotting. RESULTS ： MTT colorimetry showed that the A490 values were 0. 417±0. 011, 0. 622±0. 015 and 0. 602±0. 013, respectively in the 5μg/L, 10 μg/L and 20μg/L TNF-α groups, all significantly higher than that in the control group （0. 235±0. 013, all P 〈0.05）. After stimulation of 5μg/L and 10 μg/L TNF-α for 36 h, cyclinD1 was upregulated both in mRNA （0. 706 ±0. 113, 1. 698±0. 135 ） and protein （ 1. 270±0. 168, 2. 749±0. 170） levels compared with those in control group （0. 192±0. 039 and 0. 658±0. 101 ） （all P 〈 0.01 ）. CONCLUSION： Tumor necrosis factor-α promotes CF proliferation through upregulation of cyclinD1. Our findings may provide evidence of using TNF-α signal pathway inhibitor for anti-fibrosis.