摘要
目的 探讨Wnt信号通路调控剂分泌型卷曲相关蛋白3(sFRP3)在小鼠心肌成纤维细胞(CFs)活化增殖中的作用.方法 购入1～3 d的小鼠乳鼠,行手术对心脏取材,消化后分离CFs进行培养.细胞贴壁生长后使用转化生长因子(TGF-β1)刺激构建CFs活化增殖模型;确认模型构建成功后分别向实验组和对照组细胞转染sFRP3过表达质粒和空载质粒24～48 h.通过Western blot、qRT-PCR的方法在分子层面对sFRP3、Periostin(POSTN)、Ⅰ型胶原(Collagen Ⅰ)和增殖细胞核抗原(PCNA)的表达进行检测;使用MTT法、CCK-8法和EdU染色法检测细胞增殖能力的改变.结果 在TGF-β1刺激构建的CFs活化增殖模型中,相较于对照组,模型组sFRP3蛋白及mRNA表达下降,活化增殖相关蛋白PCNA、POSTN和Collagen Ⅰ表达上调.另外,在质粒转染sFRP3过表达组的CFs中,PCNA、POSTN和Collagen Ⅰ蛋白及mRNA表达相较于空载组下降.MTT、CCK-8与EdU实验表明,质粒转染sFRP3过表达组的CFs增殖活性较空载组明显下降.结论 过表达sFRP3明显抑制CFs活化增殖,提示sFRP3可能是参与调控CFs活化增殖的关键基因.
Abstract
Objective To explore the role of secreted frizzled-related protein 3 (sFRP3), a regulator of the Wnt signaling pathway, in the activation and proliferation of murine cardiac fibroblasts (CFs).Methods Neonatal mice aged 1-3 days were obtained for surgical procedures to collect heart tissues.After digestion, CFs were isola-ted and cultured.Transforming growth factor-beta 1 (TGF-β1) stimulation was used to induce activation and prolif-eration in CFs after they adhered to the culture dish.Once the model was confirmed, experimental and control groups were transfected with sFRP3 overexpression plasmids and empty plasmids for 24-48 hours.Expression lev-els of sFRP3, Periostin (POSTN), Type Ⅰ collagen (Collagen Ⅰ), and proliferating cell nuclear antigen (PC-NA) were assessed at the molecular level using Western blot and qRT-PCR.Changes in cell proliferation capacity were examined using MTT, CCK-8, and EdU staining methods.Results In the TGF-β1-induced activation and proliferation model of CFs, compared to the control group, the model group exhibited decreased expression of sFRP3 protein and mRNA, while the expression of activation and proliferation-related proteins PCNA, POSTN, and Collagen Ⅰ was upregulated.Furthermore, in CFs overexpressing sFRP3 through plasmid transfection, the protein and mRNA expression of PCNA, POSTN, and Collagen Ⅰ decreased compared to the empty vector group.MTT, CCK-8 , and EdU experiments indicated a significant decrease in the proliferative activity of CFs in the sFRP3 over-expression group compared to the empty vector group.Conclusion Overexpression of sFRP3 markedly inhibits the activation and proliferation of CFs, suggesting that sFRP3 may be a key gene involved in the regulation of CF acti-vation and proliferation.
基金项目
安徽省重点研究与开发计划项目(202104j0702-0037)
安徽省高校优秀青年科研项目(2023AH-030116)
安徽高校自然科学研究项目(KJ2021A0313)
国家科技期刊平台
NSTL
万方数据