CD137-CD137L信号通过CaM-CaN通路调控小鼠血管平滑肌细胞NFATc1表达

CD137-CD137L signaling regulates NFATc1 expression in mice vascular smooth muscle cells through CaM-CaN pathway

目的:探讨CD137-CD137L信号是否通过钙调蛋白-钙调神经磷酸酶(Calmodlin-Calcineurin,CaM-CaN)通路调控小鼠血管平滑肌细胞活化T细胞核因子c1(nuclear factor of activated T cells c1,NFATc1)的表达。方法:采用改良组织贴块法原代培养小鼠血管平滑肌细胞。实验分两部分,第一部分细胞分组:对照组、CD137刺激组(激动型CD137抗体10μg/m L)和CD137抑制组(抑制型CD137抗体10μg/m L预孵30 min后+激动型CD137抗体10μg/m L);第2部分细胞分组:对照组、CD137刺激组(激动型CD137抗体10μg/m L)、si CaM组(si-CaM+CD137刺激组)和si CaN组(si-CaN+CD137刺激组)。分别采用蛋白质印迹和RT-PCR检测平滑肌细胞CaM、CaN及NFATc1的蛋白和mRNA表达水平。结果:(1)蛋白质印迹和RT-PCR结果显示,与对照组相比,CD137刺激组小鼠平滑肌细胞CaM、CaN及NFATc1表达明显升高(P<0.05);与CD137刺激组相比,CD137抑制组CaM、CaN及NFATc1表达显著降低(P<0.05)。(2)通过si-RNA技术分别沉默CaM和CaN后,与CD137刺激组相比,si CaM组CaM、CaN及NFATc1表达水平明显降低(P<0.05);si CaN组的CaM无明显改变(P>0.05),而CaN及NFATc1表达明显降低(P<0.05)。结论:CD137-CD137L信号可能通过CaM-CaN通路调控NFATc1的表达。

Objective： To observe whether CD137 signaling affects the expression of nuclear factor of ac- tivated T cells cl （NFATcl） in vascular smooth muscle cells （VSMCs） through Calmodlin-Calcineurin （CaM-CaN） pathway. Methods： Isolated VSMCs from the mice aortic by explant tissue culture. The ex- periment was divided into two parts. Part I ： the prepared cells were divided into three groups： control group, CD137 activated group, CD137 inhibited group; Part ll： control group, CD137 activated group （agonist CDI37mAb）, si-CaM group （si-CaM ＋ CD137 activated group）, si-CaN group （ si-CaN ＋ CD137 activated group）. The levels of CaM, CaN and NFATcl were evaluated by Western blotting and RT-PCR, respectively. Results： （1） The protein and mRNA levels of CaM, CaN and NFATcl were increased markedly by agitating CD137-CD137L signal comparing with control group. When CD137-CD137L signaling was restrained by anti-CD137 mAb, the protein and mRNA levels of CaM, CaN and NFATcl were de- creased markedly comparing with CD137 activated group. When blocked CaM by CaM siRNA, the protein and mRNA levels of CaM, CaN and NFATcl were decreased after stimulated by agonist CD137 mAb comparing with CD137 activated group. When blocked CaN, the levels of CaN and NFATcl were decreased but the level of CaM did not change after stimulated by agonist CD137 mAb comparing with CD137 activated group. Conclusion： CD137-CD137L signaling could regulate the expression of NFATcl through CaM-CaN pathway.