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(CollagenⅠ)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CollagenⅠ表达上调。另外,在质粒转染sFRP3过表达组的CFs中,PCNA、POSTN和CollagenⅠ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。
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 isolated and cultured.Transforming growth factor-beta 1(TGF-β1)stimulation was used to induce activation and proliferation 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 levels of sFRP3,Periostin(POSTN),TypeⅠcollagen(CollagenⅠ),and proliferating cell nuclear antigen(PCNA)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 CollagenⅠwas upregulated.Furthermore,in CFs overexpressing sFRP3 through plasmid transfection,the protein and mRNA expression of PCNA,POSTN,and CollagenⅠdecreased 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 overexpression 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 activation and proliferation.
作者
江舜祥
涂彬
宋凯
何缓缓
陶辉
曹炜
Jiang Shunxiang;Tu Bin;Song Kai;He Huanhuan;Tao Hui;Cao Wei(Dept of Thoracic Surgery,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;Dept of Anesthesiology and Perioperative Medicine,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601)
出处
《安徽医科大学学报》
CAS
北大核心
2024年第5期809-814,共6页
Acta Universitatis Medicinalis Anhui
基金
安徽省重点研究与开发计划项目(编号:202104j0702-0037)
安徽省高校优秀青年科研项目(编号:2023AH-030116)
安徽高校自然科学研究项目(编号:KJ2021A0313)。
