机械刺激对心脏成纤维细胞增殖、活化、凋亡的影响

Effect of mechanical stimulation on proliferation,activation and apoptosis of cardiac fibroblasts

目的观察不同机械刺激对心脏成纤维细胞表型的影响。方法采用自主研发的双轴动态培养装置分别以3%、6%、9%的拉伸幅度和1.00、1.33、1.66 Hz的拉伸频率对乳鼠心脏成纤维细胞(CFs)进行机械刺激,以静止状态培养的CFs为对照组,免疫荧光法检测CFs增殖蛋白Ki-67的表达变化,反转录-聚合酶链式反应(RT-PCR)检测活化蛋白胶原蛋白Ⅰ(Col-Ⅰ)、胶原蛋白Ⅲ(Col-Ⅲ)和平滑肌肌动蛋白(α-SMA)与凋亡蛋白半胱氨酸蛋白酶3(Caspase-3)的表达变化。组间比较采用方差分析,各组与对照组的比较采用Dunnett-t检验。结果 RT-PCR结果显示,当拉伸幅度为6%时,与静止组比较Col-Ⅰ的表达水平上升(1.51±0.43,t=2.470,P<0.05),差异有统计学意义,当拉伸频率为1.33 Hz时,与静止组比较Col-Ⅰ表达水平的上升(1.54±0.41,t=2.771,P<0.05),差异有统计学意义,机械刺激对Col-Ⅲ、α-SMA,Caspase-3的表达变化则无明显影响;免疫荧光结果显示,机械刺激对Ki-67没有明显影响。结论当拉伸幅度为6%、拉伸频率为1.33 Hz时,机械刺激能够引起乳鼠心脏成纤维细胞活化相关蛋白Col-Ⅰ表达的上调。

Objective To observe the effects of different mechanical stimuli on the phenotype of cardiac fibroblasts(CFs).Methods The cardiac fibroblasts of neonatal mice(CFs)were mechanically stimulated by the independently developed biaxial dynamic culture device at the stretching amplitude of 3%,6%and 9%or the stretching frequency of 1.00hz,1.33hz and 1.66hz,respectively.The CFs cultured in static state was used as the control group.The expression changes of CFs proliferating protein Ki67 were detected by immunofluorescence.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression changes of activated protein collagenⅠ(Col-Ⅰ),collagenⅢ(Col-Ⅲ),α-smooth muscle actin(α-SMA)and apoptotic protein caspase3.Data were expressed as mean±standard deviation(Mean±SD),and variance analysis was used,The Dunnett-t test was used to compare each group with the control group.Results RT-PCR results showed that the expression level of Col-Ⅰincreased(1.51±0.43,t=2.470,P<0.05)when the stretching amplitude was 6%,and the expression level of Col-Ⅰincreased(1.54±0.41,t=2.771,P<0.05)when the stretching frequency was 1.33 Hz,as compared with those of the static group.Immunofluorescence results showed that mechanical stimulation had no significant effect on Ki-67.Conclusion When the stretching amplitude was 6%and the stretching frequency was 1.33 Hz,mechanical stimulation could induce the expression of Col-Ⅰ,an activation-related protein in CFs of neonatal mice.