摘要
目的探讨Wnt/β-catenin信号通路在可溶性卷曲相关蛋白1(Sfrp1)抑制乳鼠心肌纤维化中的作用及机制。方法选择日龄1~3 d的SD新生乳鼠8只,分离培养乳鼠心肌成纤维细胞,分为对照组、增殖模型组(TGF-β1组)、Sfrp1预处理组(TGF-β1+Sfrp1组)、激活剂实验组(TGF-β1+Sfrp1+Licl组)。对照组不加任何处理。TGF-β1组加入TGF-β1 10 ng/mL刺激12 h,建立增殖模型。TGF-β1+Sfrp1组先转染dsAAV9-Sfrp1病毒,第5天时加入TGF-β1 10 ng/mL刺激12 h。TGF-β1+Sfrp1+Licl组先转染dsAAV9-Sfrp1病毒,待孵育第4天时,将Wnt/β-catenin信号通路特异性激活剂Licl加入培养基,终浓度为20 mmol/L,然后加入TGF-β1 10 ng/mL。采用Western blotting法检测Wnt/β-catenin信号通路蛋白β-catenin、Dvl-1及肌成纤维细胞分化的标志蛋白α-SMA表达。采用MTT法检测细胞增殖。采用流式细胞术检测细胞周期。采用ELISA法检测细胞上清液Ⅰ型、Ⅲ型胶原含量。结果与对照组相比,TGF-β1组β-catenin、Dvl-1、α-SMA蛋白表达均上调,OD值及Ⅰ、Ⅲ型胶原蛋白含量均升高(P均<0.05);与TGF-β1组相比,TGF-β1+Sfrp1组β-catenin、Dvl-1、α-SMA蛋白表达均下调,OD值及Ⅰ、Ⅲ型胶原均降低(P均<0.05);与TGF-β1+Sfrp1组相比,TGF-β1+Sfrp1+Licl组β-catenin、Dvl-1、α-SMA蛋白表达均上调,OD值及Ⅰ、Ⅲ型胶原蛋白含量均升高,G0/G1期、G2/M期细胞百分比均降低,S期细胞百分比均升高(P均<0.05)。结论Sfrp1可通过抑制Wnt/β-catenin信号通路,有效抑制心肌成纤维细胞增殖、胶原合成,促进肌成纤维细胞表型转化,从而缓解心肌纤维化的进展。
Objective To investigate the role and relative molecular mechanism of Wnt/β-catenin signaling pathway in the process of soluble frizzled related protein1(Sfrp1)inhibiting the cardiac fibroblasts of neonatal rats.Methods Eight SD neonatal rats of 1 to 3 days old were selected,and the cultured neonatal rat cardiac fibroblasts were isolated and divided into the control group,proliferation model group(TGF-β1 group),Sfrp1 pretreatment group(TGF-β1+Sfrp1 group),and activator experimental group(TGF-β1+Sfrp1+Licl group).The control group received no treatment.The rats in the TGF-β1 group were stimulated with TGF-β1 10 ng/mL for 12 h to establish the proliferation models.The rats in the TGF-β1+Sfrp1 group were first transfected with dsAAV9-Sfrp1 virus,and on day 5,TGF-β1 was added at 10 ng/mL for 12 h.The rats in the TGF-β1+Sfrp1+Licl group were first transfected with dsAAV9-Sfrp1 virus,and on the 4th day of incubation,the Wnt/β-catenin signaling pathway specific activator Licl was added to the medium at a final concentration of 20 mmol/L,then 10 ng/mL TGF-β1 was added.The expression of Wnt/β-catenin signaling pathway proteinsβ-catenin,Dvl-1 and the marker proteinα-SMA for myofibroblast differentiation was detected by Western blotting.The cell proliferation was measured by MTT assay.Cell cycle was detected by flow cytometry.The content of typeⅠand typeⅢcollagen in the supernatant of the cells was determined by ELISA.Results Compared with the control group,the expression levels ofβ-catenin,Dvl-1 andα-SMA were up-regulated,and OD value,ⅠandⅢcollagen content increased in the TGF-β1 group(all P<0.05).Compared with the TGF-β1 group,the expression levels ofβ-catenin,Dvl-1 andα-SMA were downregulated,and OD,ⅠandⅢcollagen decreased in the TGF-β1+Sfrp1 group(all P<0.05).Compared with the TGF-β1+Sfrp1 group,the expression levels ofβ-catenin,Dvl-1 andα-SMA were up-regulated,and OD value,ⅠandⅢcollagen content increased,and the percentage of cells in the G0/G1 phase and G2/M phase decreased,and the percentage of cells in S phase increased in the TGF-β1+Sfrp1+Licl group(all P<0.05).Conclusion Sfrp1 may decrease the proliferation of cardiac fibroblasts and collagen synthesis,and promote the phenotype transformation of muscle fibroblasts by inhibiting the Wnt/β-catenin signaling pathway,and thus alleviating the development of myocardial fibrosis.
作者
陶静
魏娴
马依彤
TAO Jing;WEI Xian;MA Yitong(People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China)
出处
《山东医药》
CAS
2019年第23期10-13,共4页
Shandong Medical Journal
基金
新疆维吾尔自治区卫生健康青年医学科技人才专项(WJWY-201905)
新疆维吾尔自治区青年科学基金项目(2017D01C347)
