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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
作者:刘丹 田孝祥 刘美丽 刘艳霞 齐艳萍 陶杰 闫承慧 
单位:110016 沈阳 北部战区总医院心内科(刘丹、田孝祥、刘美丽、刘艳霞、齐艳萍、陶杰、闫承慧) 
关键词:CC类趋化因子受体2 心肌梗死 缺氧 心肌成纤维细胞 表型转换 
分类号:R541.4
出版年,卷(期):页码:2019,44(1):1-6
摘要:

[摘要]  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PCRWestern 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 Western blotting检测CCR2α-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-48hCCR2 mRNA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-CCR2α-SMACol 1A基因mRNA 和蛋白水平均无明显变化,而si-control+缺氧组CCR2α-SMACol 1A基因mRNA和蛋白水平均明显升高(P<0.01P<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-SMACol 1A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。

[Abstract]   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-24h and hypoxia-48h, the mRNA and protein expression of CCR2 was examined by real-time 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 1A (Col 1A) 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-24h and hypoxia-48h 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 Col 1A not significantly changed in si-CCR2 group. Compared with si-control group, the mRNA and protein levels of CCR2, α-SMA and Col 1A 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 1A 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.

基金项目:
国家自然科学基金面上项目(81570265、81670276、81770303);辽宁省自然科学基金(20180550368、20170540929);军事科技领域青年人才托举工程项目(17-JCJQ-QT-028);辽宁省博士启动基金(201601407)
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