目的 探讨FERM、ARH/RhoGEF和pleckstrin结构域蛋白1(Farp1)在胸主动脉夹层中的表达及作用.方法 胸主动脉夹层(TAD)组织来源于2022年12月至2023年3月在武汉大学中南医院因Stanford A型主动脉夹层行外科手术的患者,收集心脏移植供体残余的胸主动脉作为对照(CRTL)组.通过蛋白质印迹法(Western blot)和免疫组织化学检测TAD与CRTL组胸主动脉组织中Farp1的表达水平;3周龄雄性C57BL/6J小鼠喂养0.25％的β-氨基丙腈酯(BAPN)4周构建胸主动脉夹层模型,检测指标及方法同人胸主动脉组织;血管紧张素Ⅱ(AngⅡ,1×10-7 mol/L)刺激原代大鼠胸主动脉平滑肌细胞(RAVSMCs)24 h模拟胸主动脉夹层,构建Ad-sh-Farp1敲低腺病毒干扰RAVSMCs中Farp1的表达,通过Western blot检测RAVSMCs中收缩表型蛋白标志物.采用非配对t检验进行统计学分析.结果 Western blot(1.723±0.084,t=5.667,P＜0.05)及免疫组织化学染色(3.507±0.232,t=4.393,P＜0.05)结果显示人TAD组织中Farp1的蛋白水平高于对照组;小鼠主动脉夹层组织 Western blot(1.304±0.042,t=2.996,P＜0.05)和免疫组织化学染色(0.458±0.030,t=4.060,P＜0.05)显示了相同结果,且免疫组织化学染色进一步提示Farp1蛋白水平改变主要发生于主动脉中层(平滑肌);细胞实验显示Ang Ⅱ促进RAVSMCs中Farp1蛋白表达上调(1.469± 0.115,t=3.912,P＜0.05),敲低 Farp1 抑制了 RAVSMCs 表型转换[α-平滑肌肌动蛋白(α-SMA):1.762±0.202,t=3.123,P＜0.05;CNN1∶1.615±0.101,t=5.288,P＜0.05;SM22α:1.618±0.135,t=2.964,P＜0.05].结论 Farp1与主动脉平滑肌细胞表型转换明显相关,Farp1蛋白水平增高可能促进胸主动脉夹层的发生发展.
Expression and role of FERM,ARH/RhoGEF and pleckstrin domain protein 1 in thoracic aortic dissection
Objective To investigate the expression and role of FERM,ARH/RhoGEF and pleck-strin domain protein 1(FARP1)in thoracic aortic dissection(TAD).Methods Patients who underwent surgical intervention for Stanford type A aortic dissection at Zhongnan Hospital of Wuhan University from December 2022 to March 2023 were selected as the TAD group.The control(CRTL)group consisted of re-sidual thoracic aortic tissues from heart transplant donors.The expression levels of FARP1 in TAD group and CRTL group were detected by Western blotting and immunohistochemical staining.The 3-week-old male C57BL/6J mice were fed on 0.25％β-aminopropionitrile(BAPN)for 4 weeks to establish a TAD model.The detection indexes and methods were the same as those in human thoracic aortic tissues studies.Primary rat aortic vascular smooth muscle cells(RAVSMCs)were stimulated with angiotensin Ⅱ(Ang Ⅱ,1× 10-7 mol/L,24 h)to mimic TAD.Ad-sh-FARP1 adenovirus was used to knock down FARP expression in RAVSMCs,and Western blotting was performed to detect the contractile phenotype protein markers.A non-paired t-test was utilized for statistical analysis of the data.Results The results of Western blotting(1.723±0.084,t=5.667,P＜0.05)and immunohistochemical staining(3.507±0.232,t=4.393,P＜0.05)revealed significantly increased levels of FARP1 protein in human TAD tissues compared to the CRTL group.Similar results were observed in Western blotting(1.304±0.042,t=2.996,P＜0.05)and immunohistochemical staining(0.458±0.030,t=4.060,P＜0.05)of mouse aortic dissection tissues,with further indication that the change in FARP1 protein level primarily occurred in the medial layer(smooth muscle)of the aorta.Celluar experiments showed that Ang Ⅱ stimulation promoted upregulation of FARP1 protein expression(1.469±0.115,t=3.912,P＜0.05),while knocking down FARPl sup-pressed phenotypic switch in RAVSMCs[α-smooth muscle actin(α-SMA):1.762±0.202,t=3.123,P＜0.05;CNN1∶1.615±0.101,t=5.288,P＜0.05;SM22α:1.618±0.135,t=2.964,P＜0.05].Conclusion FARP1 is significantly associated with phenotypic switch of aortic smooth muscle cells,and increased levels of FARP1 protein may promote the occurrence and development of TAD.
NETL
NSTL
万方数据
