【摘要】 目的观察睾酮对心脏老化过程中心肌纤维化的干预效应。方法差速贴壁法培养雄性昆明白乳鼠心肌成纤维细胞,并分为4组:A组（培养液）、B组（60 μmol/L H₂O₂）、C组（30 nmol/L 睾酮+B组）、D组（氟他胺1μmol/L+C组）。用β半乳糖苷酶染色检测细胞的阳性率,MTT法检测细胞增殖能力,流式细胞仪分析细胞周期,二氯荧光黄荧光显色检测细胞内活性氧（ROs）。α平滑肌肌动蛋白（α-SMA）免疫化学染色反应细胞的表型转化,RT-PCR法检测转化生长因子β1（TGF-β1）mRNA、P16 mRNA和cyclin D1 mRNA的表达。结果与A组比较.B组β-半乳糖苷酶染色阳性细胞增加,ROS水平增加,P16 mRNA、cyclin D1 mRNA、TGF-β1 mRNA表达上调,细胞增殖能力明显下降,G₀/G₁期细胞比例增多,同时α-SMA免疫组织化学染色出现明显强阳性;与B组比较,C组上述各项指标下降;与C组比较,D组能部分阻断睾酮的各种保护作用。结论睾酮通过受体机制干预氧化应激诱导的心肌成纤维细胞的衰老,这可能是睾酮抗心脏老化过程中心肌纤维化的机制之一。更多还原

【Abstract】 Objective To observe the intervention effect of testosterone on the cardiac fibrosis during aging process of heart. Methods Cardiac fibroblasts from ventricle of heart of neonatal male Kunming mice were cultured using differential anchoring velocity method. The cultured cardiac fibroblasts were divided into four groups: group A （culture medium）, group B（60 μmol/L H₂O₂）, group C（group B+30 nmol/L testosterone） and group D（group C+flutamide 1 μmol/L）. The expression of SA-β-gal was detected by SA-β-gal actosidase staining and the proliferation of cells was detected by MTT method,the cell cycle was analyzed by a FACS coulter counter,α-SMA level was determined by immunochemical staining, 2,7-dichlorofluorescein diacetate was used to determine intracellular ROS. The changes of expression of TGF-β1,P16 and cyclin D1 mRNA were measured by RT-PCR. Results Compared with group A, H₂O₂ caused more cellular senescence and intracellular ROS,and up-regulation of P16 mRNA,cyclin D1 mRNA and TGF-β₁ mRNA expression, which induced G₁ cell cycle arrest and inhibited the cell proliferation. Furthermore, the expression of α-SMA was stimulated while TGF-β₁ mRNA expression was upregulated. Testosterone pretreatment significantly attenuated all these effects of H₂O₂ exposure. Flutamide administered prior to testosterone partly antagonized the protective effects of testosterone against H₂O₂ exposure. Conclusion The anti-senescence effects of testosterone on cardiac fibroblasts might be one of mechanisms for fighting against cardiac fibrosis during aging of heart.更多还原