摘要
目的研究ERK1/2通路在血小板源性生长因子(PDGF)-CC诱导的鼠心肌纤维化中的作用及其可能的机制。方法取SD大鼠乳鼠心脏组织,差速贴壁法分离并纯化心肌成纤维细胞。按不同药物处理随机分成对照组(CON组)、PDGF-CC(P组)、PDGF-CC+ERK1/2抑制剂U0126(PU组)。MTT法检测心肌成纤维细胞的增殖,qRT-PCR法检测mRNA含量,Western blot法分析蛋白表达量。结果MTT法显示P组细胞数较CON组显著增加(P<0.01),而PU组细胞数较P组明显减少(P<0.01)。qRT-PCR法显示P组中PDGF-α受体(PDGFR-α)、ERK1、ERK2、Ⅰ型和Ⅲ型胶原蛋白(ColⅠ、ColⅢ)的mRNA表达水平均明显高于CON组(P<0.001),PDGFR-β的mRNA表达量在P组与CON组间差异无统计学意义。与P组相比,PU组中PDGFR-α、ERK1、ERK2、ColⅠ和ColⅢmRNA表达均明显下降(P<0.01)。Western blot法显示P组中磷酸化PDGFR-α(pPDGFR-α)、ERK1/2、p-ERK1/2、ColⅠ和ColⅢ的表达量均明显高于CON组(P<0.001),PU组中p-PDGFR-α、ERK1/2、p-ERK1/2、ColⅠ和ColⅢ蛋白表达量均显著低于P组(P<0.001)。结论 PDGF-CC可能通过结合PDGFR-α激活ERK1/2信号通路,诱导鼠心肌成纤维细胞过量增殖伴胶原蛋白的合成,参与心肌纤维化的发生。
Objective To investigate the role and mechanism of ERK1/2 pathway in PDGF-CC-induced cardiac tl- brosis in rats. Methods The cardiac fibroblasts were isolated and purified from cardiac tissues of SD neonatal rats by differential time attachment,then divided into 3 groups randomly : control group ( CON), PDGF-CC group (P) and group of PDGF-CC + ERK1/2 inhibitor U0126(PU). MTT assay was used to detect the proliferation of cardiac fibroblasts. The mRNA expression levels were detected by real-time fluorescent quantitative PCR (qRT-PCR). The protein expressions were detected by Western blot. Results MTT showed that the number of cells in P group was significantly higher than that in CON group(P 〈 0.01 ) , while the number of cells in PU group was significandy lower than that in P group ( P 〈 0.01 ). qRT-PCR showed that the mRNA expressions of PDGFR-α( PDGF receptor- α), ERK1, ERK2, collagen type I and type Ⅲ (Col I ,Col Ⅲ) were significantly higher than CON group(P 〈 0. 001 ) , PDGFR-β mRNA expression showed no significant difference between P and CON group, and that PDG- FR-α,ERK1 ,ERK2,Col I and Col m in PU group was markedly reduced as compared to P group (P 〈 0. 01 ). Western blot showed that the protein expressions of phosphorylated PDGFR-α( p-PDGFR-α), ERK1/2, p-ERK1/ 2, Col I and Col IR were significantly higher than that in CON group(P 〈 0. 001 ) , and those expressions were significantly decreased in the PU group compared with P group (P 〈 0. 001 ). Conclusion PDGF-CC may activate the ERK1/2 signaling pathway by binding to PDGFR-α, leads to excessive proliferation of rat cardiac fibroblasts with collagen synthesis and participate in the development of myocardial fibrosis.
出处
《安徽医科大学学报》
CAS
北大核心
2017年第10期1489-1494,共6页
Acta Universitatis Medicinalis Anhui
基金
安徽省科技攻关计划项目(编号:1604a0802074)
安徽省公益性技术应用研究联动计划项目(编号:15011d04032)