CD137信号通过外泌体传递活化T细胞核因子c1介导小鼠血管平滑肌细胞钙化
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(江苏大学附属医院心内科,江苏省镇江市 212001)

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王宁,硕士研究生,研究方向为心血管疾病及动脉粥样硬化,E-mail为769353653@qq.com。通信作者严金川,博士,教授,研究方向为心血管疾病及动脉粥样硬化,E-mail为yanjinchuan@hotmail.com。

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国家自然科学基金(81670405);江苏省自然基金(BK20161355);江苏省研究生科研创新计划(KYCX18_2286);镇江市心血管病临床医学研究中心(SS2018008)


CD137 signal mediates calcification of mouse vascular smooth muscle cells via exosomes transmitting nuclear factor of active T cell c1
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Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001, China)

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    摘要:

    目的 探讨外泌体在CD137信号诱导的小鼠血管平滑肌细胞(VSMC)钙化中的作用。方法 组织贴壁法提取小鼠胸主动脉VSMC,将细胞分为2组,即对照组和CD137激动组,用试剂盒提取外泌体,并用透射电镜、纳米颗粒跟踪分析法及Western blot鉴定和分析,荧光显微镜观察VSMC摄取外泌体。构建活化T细胞核因子c1(sh-NFATc1)慢病毒载体并感染VSMC。实验分为3组:对照组外泌体处理组、CD137激动组外泌体处理组、沉默NFATc1+CD137激动组外泌体处理组。采用Western blot检测α平滑肌肌动蛋白(α-SMA)、骨形成蛋白2(BMP-2)、Runt相关转录因子2(Runx-2)蛋白表达量。Von Kossa染色检测VSMC内钙盐沉积情况。结果 Western blot结果显示,2组微囊泡均表达外泌体表面标记蛋白CD9、CD81;电镜下外泌体成圆形和杯托状,直径在30~100 nm;CD137激动组外泌体中NFATc1蛋白表达显著增多。与对照组外泌体处理组比较,CD137激动组外泌体处理组钙化相关指标BMP-2、Runx-2蛋白表达显著增高,同时α-SMA表达显著下降;与CD137激动组外泌体处理组比较,沉默NFATc1+CD137激动组外泌体处理组BMP-2、Runx-2蛋白表达显著减少,α-SMA表达显著增高。Von Kossa染色结果显示,CD137激动组外泌体处理组VSMC钙盐沉积多于对照组外泌体处理组,而沉默NFATc1+CD137激动组外泌体处理组钙盐沉积比CD137激动组外泌体处理组显著降低。结论 CD137信号通路通过外泌体转运NFATc1介导VSMC钙化。

    Abstract:

    Aim To investigate the role of exosomes in the calcification of mouse vascular smooth muscle cells (VSMCs) induced by CD137 signal. Methods The mouse thoracic aorta VSMCs were performed by patch-attaching method, and VSMCs were divided into two groups:the control group, the CD137 excitation group. The exosomes were extracted by kit and identified by transmission electron microscopy, nanoparticle tracking analysis and Western blot. Fluorescence microscopy was used to observe VSMCs uptake of exosomes. Lentiviral vector of nuclear factor of active T cell c1 (sh-NFATc1) was constructed and infected with VSMCs. The experiments were divided into three groups:control group with exosomes treatment, CD137 excitation group with exosomes treatment, silenced NFATc1+CD137 excitation group with exosomes treatment. Western blot was used to detect the expressions of α-smooth muscle actin (α-SMA), bone morphogenic protein 2 (BMP-2) and Runt-related transcription factor 2 (Runx-2). Calcium salt deposition in VSMCs was detected by Von Kossa staining. Results Western blot results showed that microvesicles in both groups expressed exosomes surface marker proteins CD9 and CD81. Under electron microscopy, the exosomes were round and cup-shaped, and its diameter was about 30-100 nm. The expression of NFATc1 protein increased significantly in exosomes of CD137 excitation group. Compared with the control group exosomes treatment group, the expressions of calcification-related indicators BMP-2 and Runx-2 proteins increased significantly in CD137 excitation group with exosomes treatment, while the expression of α-SMA decreased significantly. Compared with CD137 excitation group with exosomes treatment, the expressions of BMP-2 and Runx-2 proteins decreased significantly in silenced NFATc1+CD137 excitation group with exosomes treatment, while the expression of α-SMA increased significantly. Von Kossa staining showed that VSMCs calcium deposits in CD137 excitation group with exosomes treatment were more than those in control group with exosomes treatment, while those in silenced NFATc1+CD137 excitation group with exosomes treatment were significantly lower than those in CD137 excitation group with exosomes treatment. Conclusion CD137 signal pathway mediates VSMCs calcification through exosome transmitting NFATc1.

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王宁,崔星钢,杨萍,许尧,李波,仲威,邵晨,王中群,严金川. CD137信号通过外泌体传递活化T细胞核因子c1介导小鼠血管平滑肌细胞钙化[J].中国动脉硬化杂志,2018,26(12):1194~1200, 1244.

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  • 收稿日期:2018-10-13
  • 最后修改日期:2018-11-19
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  • 在线发布日期: 2018-12-27