CD137-CD137配体信号通路通过P38调控小鼠血管平滑肌细胞钙化形成  

CD137-CD137L interaction induced the calcification of mouse smooth muscle cells via P38 MAPK signaling

作　　者：丁亮[1] 许尧 杨萍[1] 陈蕊[1] 李波[1] 邵晨[1] 仲威[1] 王中群[1] 严金川[1] Ding Liang;Xu Yao;Yang Ping;Chen Rui;Li Bo;Shao Chen;Zhong Wei;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11期892-900,共9页Chinese Journal of Cardiology

基　　金：国家自然科学基金(81670405,81370409);江苏省自然基金(BK20161355).

摘　　要：目的 探讨CD137-CD137配体(CD137L)信号通路是否通过P38调控小鼠血管平滑肌细胞(VSMC)钙化的形成.方法 C57雄性小鼠,8周龄,采用组织贴块法培养原代小鼠VSMC,取第3~8代细胞进行实验.将细胞分为4组,即对照组、CD137激动组(重组CD137L干预)、P38抑制组(CD137激动组干预基础上加P38抑制剂)和P38单独抑制组(P38抑制剂干预).采用10 mmol/L β-甘油磷酸钠+10-8mol/L地塞米松+10-7mol/L胰岛素诱导细胞钙化.免疫荧光法检测VSMC α-平滑肌肌动蛋白(α-SMA)和骨桥蛋白(OPN)的表达,Western blot法检测磷酸化P38(p-P38)、OPN、Runt相关转录因子-2(RUNX-2)蛋白表达,实时荧光定量聚合酶链反应(RT-PCR)检测钙化相关蛋白RUNX-2、OPN mRNA表达,微板法检测碱性磷酸酶(ALP)活性和钙离子浓度.进一步将细胞分为5组,即对照组、CD137激动组(重组CD137L干预)、P38抑制组(CD137激动组干预的基础上加P38抑制剂)、CD137抑制组(CD137激动组基础上加CD137抑制剂)和P38激动组(CD137抑制组的基础上加P38激动剂),通过Von Kossa染色和茜素红染色观察各组细胞的钙化程度.结果(1)各组VSMC中α-SMA和OPN蛋白表达的免疫荧光检测结果:CD137激动组VSMC中α-SMA蛋白表达水平明显低于对照组(2.79±0.25比5.42±0.47,P<0.05),而OPN蛋白表达水平则明显高于对照组(4.91±0.23比1.63±0.26,P<0.05).P38抑制组VSMC中α-SMA蛋白表达水平虽高于CD137激动组(4.48±0.27比2.79±0.25,P<0.05),但仍明显低于对照组(4.48±0.27比5.42±0.47,P<0.05);OPN蛋白表达水平虽低于CD137激动组(2.66±0.15比4.91±0.23,P<0.05),但仍高于对照组(2.66±0.15比1.63±0.26,P<0.05).而P38单独抑制组α-SMA和OPN蛋白表达水平与对照组比较差异则均无统计学意义(P均>0.05).(2)各组VSMC中p-P38、OPN、RUNX-2蛋白表达的Western blot检测结果:CD137激动组VSMC中p-P38、OPN和RUNX-2蛋白表达水平均高于对照组(分别为4.15±0.24比3.48±0.26,2.43±0.21比1.53±0.08,和3.20±0.23比1.13±0Objective To explore whether CD137-CD137L interaction could induce mouse vascular smooth muscle cells(VSMCs) calcification via P38 MAPK signaling. Methods (1) Mouse VSMCs obtained from 8-week old male C57 mice were cultured by using method of tissue piece inoculation.The cells from 3 to 8 passage were divided into 4 groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group(agonist-CD137 group+P38 inhibitor), single anti-P38 group(P38 inhibitor). The calcification was induced by adding a mixture of 10 mmol/L β-glycerophosphate+10-8mol/L dexamethasone+10-7mol/L insulin in the culture medium.Immunofluorescence was used to observe the changes of VSMCs markers(α-SMA and OPN).Real time-PCR was used to observe the mRNA expression of OPN and RUNX-2. Western blot was used to observe the protein expression of p-P38, OPN and RUNX-2. The level of cell calcification was observed by detecting alkaline phosphatase activity and calcium concentration. (2) The degeree of local calcium deposition was also tested on Von Kossa staining and Alizarin red staining methods in following 5 mouse VSMCs groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group (agonist-CD137 group+P38 inhibitor), anti-CD137 group (agonist-CD137 group +CD137 inhibitor),agonist-P38 group(anti-CD137 group+P38 agonist).Results (1) Compared with the control group, the fluorescence intensity of α-SMA was lower in the agonist-CD137 group(2.79±0.25 vs. 5.42±0.47,P<0.05), while the fluorescence intensity of OPN was higher(4.91 ± 0.23 vs. 1.63 ± 0.26, P<0.05). The fluorescence intensity of α-SMA was partly recovered after adding P38 inhibitor(4.48±0.27 vs. 2.79±0.25,P<0.05),but it was still lower than the control group(4.48±0.27 vs. 5.42±0.47,P<0.05),the fluorescence intensity of OPN decreased(2.66±0.15 vs. 4.91±0.23,P<0.05),but it was still higher than that in the control group (2.66±0.15 vs. 1.63±0.26,P<0.05).The fluorescence intensity of α-SMA and OPN(5.32±0.67 vs. 5.42±0.47,1.82±0.30 vs.

关 键 词：动脉粥样硬化 CD137 

分 类 号：R543[医药卫生—心血管疾病]