CD137-CD137L信号通路通过Rab7分子调控自噬影响小鼠血管平滑肌细胞外泌体分泌  

Impact of CD137-CD137L signaling on secretion of mouse vascular smooth muscle cells-derived exosomes: role of Rab7 pathway

作　　者：何阳 崔星刚 李波[1] 王宁 许尧 徐煜 耿田欣 严金川[1] He Yang;Cui Xinggang;Li Bo;Wang Ning;Xu Yao;Xu Yu;Geng Tianxin;Yan Jinchuan(Department of Cardiology,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,China)

机构地区：[1]江苏大学附属医院心内科,镇江212001

出　　处：《中华心血管病杂志》2019年第10期829-835,共7页Chinese Journal of Cardiology

基　　金：国家自然科学基金(81670405);江苏省自然基金(BK20161355);镇江市创新能力建设项目(SS2018008)。

摘　　要：目的 探讨CD137-CD137配体(CD137L)信号通路是否通过Rab7分子调控自噬从而影响小鼠血管平滑肌细胞(VSMC)外泌体的分泌.方法 采用组织块贴壁法原代培养C57BL/6J小鼠胸主动脉平滑肌细胞,取分离培养的第3~5代的VSMC进行实验.实验分为4组,即对照组、CD137激动组(采用10 μg/ml的CD137L重组蛋白激动CD137-CD137L信号通路)、慢病毒对照组(在CD137L重组蛋白干预的基础上加对照干扰慢病毒干预)和Rab7慢病毒干扰组(在CD137L重组蛋白干预的基础上加Rab7干扰慢病毒干预).Western blot法检测各组VSMC微管相关蛋白1轻链3(LC3Ⅱ)、p62和Rab7的蛋白表达.自噬双标腺病毒(mRFP-GFP-LC3)荧光显微镜下观察各组VSMC自噬流的变化.透射电镜下观察VSMC来源外泌体的形态及大小,Western blot法检测外泌体标记物CD9、CD81和热激同源蛋白70(Hsc70)以及自噬相关蛋白LC3Ⅱ的表达,纳米颗粒跟踪分析(NTA)技术检测各组VSMC分泌的外泌体的浓度.结果(1)各组VSMC中Rab7、LC3Ⅱ和P62的蛋白表达水平:CD137激动组VSMC中Rab7、LC3Ⅱ和p62蛋白表达水平均明显高于对照组(P均<0.05).Rab7慢病毒干扰组VSMC中Rab7、LC3Ⅱ和p62蛋白表达均低于CD137激动组(P均<0.05).慢病毒对照组VSMC中Rab7、LC3Ⅱ和p62蛋白表达与CD137激动组比较差异均无统计学意义(P均>0.05).(2)各组VSMC自噬流的变化:CD137激动组VSMC内荧光斑点总数和黄色荧光点数均多于对照组(P均<0.05),且CD137激动组VSMC内黄色荧光点数多于红色[分别为(50.3±0.9)和(10.3±1.5)个/细胞].而Rab7慢病毒干扰组VSMC内荧光斑点总数和黄色荧光点数均少于CD137激动组(P均<0.05),且Rab7慢病毒干扰组VSMC内红色荧光斑点数多于黄色[分别为(40.7±4.0)和(10.7±1.2)个/细胞].慢病毒对照组VSMC内荧光斑点总数和黄色荧光点数与CD137激动组比较差异均无统计学意义(P均>0.05).(3)小鼠VSMC来源的外泌体的鉴定、外泌体标记物CD9、CD81的表达以及各�Objective To investigate whether CD137-CD137L signaling could affect the secretion of mouse vascular smooth muscle cells (VSMCs)-derived exosomes through autophagy mediated Rab7 pathway. Methods Primary thoracic aorta VSMCs from C57BL/6J mouse were obtained by tissue block adherence method. VSMCs between the third to fifth passages were used and VSMCs were divided into 4 groups: control group, CD137 agonist group, lentivirus control group, Rab7 lentiviral interference group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 μg/ml), VSMCs in lentivirus control group were treated with lentiviral followed by recombinant protein of CD137L (10 μg/ml), VSMCs in Rab7 lentiviral interference group were treated with Rab7 lentiviral intervention followed by recombinant protein of CD137L (10 μg/ml). Western blot was used to detect the protein expression of LC3Ⅱ, p62, Rab7, CD9, CD81 and Hsc70. Fluorescence microscopy was used to track the changes of autophagy in cells infected with mRFP-GFP-LC3. Transmission electron microscope was used to observe the morphology and size of VSMCs-derived exosomes. The nanoparticle tracking analysis(NTA) was used to detect the concentration and size of exosomes in each group. Results (1) The expressions of Rab7, LC3Ⅱand p62 protein in VSMCs of CD137 activation group were significantly higher than those in control group (all P<0.05). The expressions of Rab7, LC3Ⅱ and p62 protein in Rab7 lentivirus interference group was lower than in CD137 activation group (all P<0.05), while the expressions were similar between the lentivirus control group and the CD137 activation group (all P>0.05). (2) The total number of fluorescent spots and yellow fluorescent spots in the VSMCs of the CD137 activation group were higher than those in the control group (all P<0.05), and the number of yellow fluorescent spots was higher than that of the red fluorescent spots in the VSMCs of the CD137 activation group ((50.3 ± 0.9) vs. (10.3 ± 1.5)/cell). The total numbers of fluore

关 键 词：血管 肌细胞 平滑肌 外泌体 自噬 CD137 Rab7 

分 类 号：R[医药卫生]