CC类趋化因子受体2对缺氧后小鼠心肌成纤维细胞表型转换的影响

Effects of C-C motif chemokine receptor 2 on phenotypic transformation of cardiac fibroblastsafter hypoxia

目的探讨CC类趋化因子受体2(CCR2)对缺氧后心肌成纤维细胞表型转换的影响。方法采用胶原酶消化法提取小鼠心肌原代成纤维细胞,将细胞分为对照组、缺氧-24h组和缺氧-48h组,通过定量PCR和Western blotting检测CCR2 mRNA及蛋白表达水平来确定缺氧条件。应用小干扰RNA方法建立CCR2低表达的心肌原代成纤维细胞(si-CCR2),之后将细胞分为转染对照组(si-control)、si-CCR2组、si-control+缺氧组、si-CCR2+缺氧组。应用定量PCR和Western blotting检测CCR2、α-平滑肌肌动蛋白(α-SMA)和胶原1(Col1A)表达;采用CCK8法和5-溴脱氧尿嘧啶核苷(BrdU)检测细胞增殖情况。结果与对照组比较,缺氧-24h组和缺氧-48h组CCR2mRNA及蛋白表达水平均明显升高(P<0.01),但两个时间点之间差异无统计学意义(P>0.05)。与si-control组相比,si-CCR2组α-SMA和Col1A基因mRNA和蛋白水平均无明显变化,而si-control+缺氧组CCR2、α-SMA和Col 1A基因mRNA和蛋白水平均明显升高(P<0.01或P<0.05),细胞增殖明显增加(P<0.01);与si-control+缺氧组相比,si-CCR2+缺氧组α-SMA和Col1A蛋白表达明显下降(P<0.05),细胞增殖明显降低(P<0.05)。结论 CCR2可影响缺氧后心肌成纤维细胞的表型转换。

Objective To investigate the effect of C-C motif chemokine receptor 2(CCR2) on phenotypic transformation of cardiac fibroblasts after hypoxia. Methods The mouse myocardium primary fibroblasts were extracted by collagenase digestion.Cells were divided into control, hypoxia-24 h and hypoxia-48 h, the mRNA and protein expression of CCR2 was examined by realtime PCR and Western blotting. CCR2 low expression cell(si-CCR2) was established using by small interfering RNA. Cells were divided into four groups including si-control, si-CCR2, si-control+hypoxia, si-CCR2+hypoxia. The mRNA and protein expressions of CCR2, α smooth muscle actin(α-SMA) and Collagen 1 A(Col 1 A) were detected by real-time PCR and Western blotting, cell proliferation was detected by Cell Counting Kit-8(CCK8) and Bromodeoxyuridine(BrdU). Results Compared with control, the mRNA and protein levels of CCR2 significantly increased in hypoxia-24 h and hypoxia-48 h group(P<0.01), however, no significance was found in these two time points. Compared with si-control group, the mRNA and protein expressions of CCR2, α-SMA and Col1 A not significantly changed in si-CCR2 group. Compared with si-control group, the mRNA and protein levels of CCR2, α-SMA and Col 1 A significantly increased in si-control+hypoxia group(P<0.01 or P<0.05), and cell proliferation increased(P<0.01). Compared with si-control+hypoxia group, the protein expression of α-SMA and Col 1 A decreased in si-CCR2+hypoxia group(P<0.05),and cell proliferation also decreased in si-CCR2+hypoxia group(P<0.05). Conclusions CCR2 could affect the phenotypic transformation of cardiac fibroblasts after hypoxia.