头帕肿瘤综合征蛋白在血管新生内膜形成及血管重构中的作用  

Effects of cylindromatosis on vascular neointima formation and vascular remodeling

作　　者：杨光[1] 王向东 李思颖 YANG Guang;WANG Xiangdong;LI Siying(Department of Joint Surgery,Shandong Provincial Hospital Affiliated to Shandong University,Jinan 250021,Shandong,China;Department of Physiology&Pathophysiology,School of Basic Medical Sciences,Shandong University,Jinan 250012,Shandong,China)

机构地区：[1]山东大学附属省立医院骨关节外科,山东济南250021 [2]山东大学基础医学院生理与病理生理学系,山东济南250012

出　　处：《山东大学学报（医学版）》2019年第12期37-45,共9页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81800353);山东大学基本科研业务费专项资金(2017GN0016)

摘　　要：目的探讨去泛素化酶头帕肿瘤综合征蛋白(CYLD)在血管新生内膜增生和血管平滑肌细胞表型(增殖、凋亡、炎症和分化)转化中的作用。方法利用Cyld基因敲除(Cyld^-/-)小鼠,建立血管损伤(颈总动脉结扎)模型,通过病理染色及免疫组化检测血管新生内膜的形成(H&E、Van Gieson)、细胞增殖(Ki67)、凋亡(TUNEL)以及分化簇3(CD3)、单核细胞趋化蛋白1(MCP-1)的表达;通过qRT-PCR法检测颈动脉组织中炎性细胞因子如肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、白介素6(IL-6)和抗炎细胞因子白介素10(IL-10)的表达。提取Cyld^-/-小鼠的主动脉血管平滑肌细胞(VSMCs),利用免疫荧光验证CYLD的表达,并通过生长曲线和[3H]胸腺嘧啶摄取实验观察VSMCs的生长速度,通过单核细胞黏附实验观察TNFɑ作用下VSMCs对单核细胞(THP1)的黏附作用,采用Western blotting法检测VSMCs中表型相关蛋白α平滑肌肌动蛋白(α-SMA)和平滑肌22α(SM22α)的表达。结果颈总动脉结扎4周后,Cyld^-/-小鼠的血管新生内膜增生及细胞增殖程度明显高于野生型(WT)小鼠(P<0.01);而新生内膜中细胞凋亡无明显差别。Cyld^-/-小鼠血管新生内膜中CD3、MCP-1表达及促炎因子TNFɑ、IL-1β和IL-6表达高于WT小鼠,尤其是IL-6水平显著增加(P<0.01),而抗炎因子IL-10的表达Cyld^-/-小鼠明显低于WT小鼠(P<0.01)。CYLD在Cyld^-/-小鼠的VSMCs传代过程中持续低表达。Cyld^-/-小鼠VSMCs的细胞增殖速度明显高于WT小鼠(P<0.05);在TNFɑ作用下,Cyld^-/-小鼠VSMCs对THP-1的黏附作用明显高于WT小鼠(P<0.05);Cyld^-/-小鼠VSMCs中表型相关蛋白α-SMA和SM22α的表达明显低于WT小鼠。结论在血管损伤过程中,去泛素化酶CYLD通过影响血管平滑肌细胞表型(增殖、凋亡、炎症和分化)转化,参与血管新生内膜的形成,从而在血管修复及重构中发挥关键作用。Objective To explore the role of cylindromatosis(CYLD) in vascular neointima formation and vascular smooth muscle cells phenotype transformation. Methods Cyld KO(Cyld^-/-) mice were used to establish vascular injury models of carotid artery ligation. The neointima formation was observed with H&E and Van Gieson stainings. The cell apoptosis was examined with TUNEL staining, and cell proliferation and inflammation were detected with Ki67, cluster of differentiation 3(CD3) and monocyte chemoattractant protein-1(MCP-1) immunohistiochemistry, respectively. The mRNA expressions of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) and interleukin-10(IL-10) were determined with qRT-PCR. Vascular smooth muscle cells(VSMCs) of the aortic arterywere isolated from Cyld^-/- mice. The CYLD expression was examined with immunofluorescence. The growth rate of VSMCs was observed with growth curve and 3H-thymidine intake assay. The effect of THP1 cells adhesion on VSMCs after TNF-ɑ stimulation was tested with monocyte adhesion assay. The expressions of alpha smooth muscle actin(α-SMA) and smooth muscle 22 alpha(SM22α) were measured with Western blotting. Results After carotid artery ligation for 4 weeks, the neointima formation and cell proliferation were more obvious in Cyld^-/- mice than in wild type(WT) mice(P<0.01), while there was no significant difference in cell apoptosis. The expressions of CD3, MCP-1, TNF-ɑ, IL-1β and especially IL-6 were significantly higher in Cyld^-/- mice than in WT mice(P<0.01), while the expression of IL-10 was significantly lower(P<0.01). CYLD had a low expression rate in cultured VSMCs. The proliferation rate of VSMCs was much higher in Cyld^-/- mice than in WT mice. The adhesion effect of THP-1 was more obvious in Cyld^-/- mice than in WT mice under TNF-ɑ stimulation(P<0.05). The expressions of α-SMA and SM22α were lower in Cyld^-/- mice than in WT mice. Conclusion In vascular injury, CYLD is involved in vascular neointima formation by affecting vascular smooth

关 键 词：头帕肿瘤综合征蛋白 血管平滑肌细胞 表型转化 新生内膜 血管重构 

分 类 号：R365[医药卫生—病理学]