CD137-CD137配体信号通路通过JNK途径影响小鼠血管平滑肌细胞自噬  

CD137-CD137L signaling influences the autophagy via JNK pathway in mouse vascular smooth muscle cells

作　　者：许尧 陈蕊[1] 丁亮[1] 仲威[1] 杨萍[1] 李波[1] 邵晨[1] 王中群[1] 严金川[1] Xu Yao;Chen Rui;Ding Liang;Zhong Wei;Yang Ping;Li Bo;Shao Chen;Wang Zhongqun;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》2018年第5期370-375,共6页Chinese Journal of Cardiology

基　　金：国家自然科学基金（81370409,81670405）;江苏省自然基金（BK20161355）;江苏省研究生实践创新计划（SJCX17_0585）

摘　　要：目的 探讨CD137-CD137配体（CD137L）信号通路是否通过c-Jun氨基末端激酶（JNK）途径影响小鼠血管平滑肌细胞（VSMC）自噬.方法 采用组织块贴壁法进行C57BL/6J小鼠胸主动脉VSMC原代培养,取分离培养的第3-5代VSMC进行实验.实验分为4组,即对照组、CD137激动组[加入CD137L重组蛋白（终浓度10 μg/ml）]、JNK抑制组[予JNK抑制剂SP600125（DMSO溶解,终浓度10 μmol/L）预处理细胞30 min,然后再加入CD137L重组蛋白（终浓度10 μg/ml）]和DMSO组[加入与JNK抑制组等量二甲基亚砜（DMSO）预处理30 min,然后加入CD137L重组蛋白（终浓度10 μg/ml）].Western blot法检测各组VSMC磷酸化JNK（p-JNK）、微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）及p62蛋白的表达水平.自噬双标腺病毒（mRFP-GFP-LC3）荧光显微镜观察各组VSMC自噬流变化.透射电镜观察各组VSMC内自噬小体及自噬溶酶体变化.结果 （1）各组VSMC p-JNK、LC3Ⅱ和p62蛋白表达水平：CD137激动组VSMC p-JNK、LC3Ⅱ和p62蛋白表达水平均明显高于对照组（分别为1.15±0.19比0.72：±0.21、1.03±0.13比0.59±0.15、1.10±0.19比0.76±0.15,P均〈0.05）.JNK抑制组p-JNK、LC3Ⅱ和p62蛋白表达水平均低于CD137激动组（分别为0.61±0.21比1.15±0.19、0.74±0.11比1.03±0.13、0.21±0.12比1.10±0.19,P均〈0.05）.DMSO组p-JNK、LC3Ⅱ和p62蛋白表达水平与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：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[分别为（93.00±14.11）个/细胞比（52.33±9.61）个/细胞和（64.33±6.81）个/细胞比（25.67±3.51）个/细胞,P均〈0.05],且CD137激动组VSMC内黄色荧光斑点数多于红色[分别为（64.33±6.81）、（28.67±7.51）个/细胞].而JNK抑制组VSMC内光斑点总数和黄色荧光点数均少于CD137激动组[分别为（53.00±3.17）个/细胞比（93.00±14.11）个/细胞、（15.33±4.51）个/细胞比（64.33±6.81�Objective To investigate whether CD137-CD137L signaling can affect the autophagy of mouse vascular smooth muscle cells（VSMCs） through JNK signal pathway.Methods Primary culture of C57BL/6J mouse thoracic aorta VSMCs was performed by tissue block adherence method.VSMCs between the third to fifth passages were isolated and cultured.VSMCs were divided into 4 groups：control group,CD137 agonist group,JNK inhibition group,and DMSO group,VSMCs in CD137 agonist group were treated with recombinant protein of CD137L （10 μg/ml）,VSMCs in JNK inhibition group were treated with JNK inhibitor SP600125 （10 μmol/L） for 30 minutes followed by recombinant protein of CD137L （10 μg/ml） and DMSO group was treated with the same amount of DMSO in JNK inhibition group for 30 minutes,then added recombinant protein of CD137L （10 μg/ml）.Western blot was used to detect the protein expression of p-JNK,LC Ⅱ and p62 in each group.Fluorescence microscopy was used to track the changes of autophagy in cells which was infected with adenovirus expressing tandem mRFP-GFP-LC3.Transmission electron microscope （TEM） was used to observe intracellular autophagosomes and autolysosomes.Results （1） Compared with the control group,stimulating CD137-CD137L axis by recombinant protein of CD137L significantly upregulated the expression of p-JNK,LC Ⅱ and p62 （1.15±0.19 vs.0.72±0.21,P〈0.05;1.03±0.13 vs.0.59±0.15,P〈0.05,and 1.10±0.19 vs.0.76±0.15,P〈0.05）.These effects could be reduced by JNK inhibitor （0.61±0.21 vs.1.15±0.19,P〈0.05;0.74±0.11 vs.1.03±0.13,P〈0.05,and 0.21±0.12 vs.1.10±0.19,P〈0.05）.The expression of these proteins in DMSO group remained unchanged compared with CD137 agonist group （P〉0.05）.（2） Changes of autophagy in cells of various group：the number of total fluorescent spots and yellow fluorescent spots in CD137 agonist group was significantly increased compared to control group （total fluorescent spots：（93.00± 14.11）/cell vs.（52.33 ± 9.61）/cell,P〈0.05,a

关 键 词：动脉粥样硬化 血管 肌细胞 平滑肌 抗原 CD137 自噬 

分 类 号：R543.5[医药卫生—心血管疾病]