【摘要】 目的探究蛋白激酶Cα-核因子κB(PKCα-NF-κB)级联对哮喘血清被动致敏的人气道平滑肌细胞周期蛋白D1(Cyclin D1)的调节作用及细胞增殖的影响。方法用10%的哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs),予以蛋白激酶C(PKC)激活剂12-肉豆蔻酰-13-乙酸佛波酯(PMA)刺激。分别以PΚCα反义寡核苷酸(PΚCα-asODN)和吡咯烷二硫氨基甲酸(PDTC)抑制PKCα表达和NF-κB活化。用凝胶电泳迁移率改变试验(EMSA)检测HASMCs中NF-κB的活性,用RT-PCR法及Western blot法检测干预前后Cyclin D1的mRNA及蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MTT)法检测HASMCs增殖。结果PMA刺激后磷酸化PKCα(p-PKCα)水平增高,NF-κB-DNA结合活性增强,Cyclin D1表达明显增强,HASMCs的增殖增强;而反义PKCα寡核苷酸转入细胞特异性地抑制PΚCα表达后,p-PKCα水平下降,NF-κB-DNA结合活性明显减弱,Cyclin D1表达也明显下降,HASMCs的增殖减弱(P<0.05,n=4);用PDTC抑制NF-κB活性后,PMA刺激下p-PΚCα水平仍然明显增高,但Cyclin D1的表达明显下降,HASMCs的增殖减弱(P<0.05,n=4)。结论NF-κB是PKCα的下游信号分子,PΚCα-NF-κB级联参与了PMA所诱导的哮喘血清被动致敏的人气道平滑肌细胞Cyclin D1的表达上调及细胞增殖。更多还原

【Abstract】 Objective To explore the role of PΚCα-NF-κB cascade in the PMA-induced up-regulation of Cyclin D1 in atopic asthmatic sensitized human airway smooth muscle cells (HASMCs). Methods HASMCs in culture were passively sensitized with 10% serum from asthmatic patients,and stimulated with PΚC activator PMA. The expression of PΚCα and NF-κB activity were inhibited with PΚCα antisense oligodeoxynucleotides (PΚCα-asODN) and PDTC respectively. The NF-κB activity of HASMCs was analyzed by electrophoretic mobility gel shift assay (EMSA). The expression levels of Cyclin D1 mRNA and protein were detected by RT-PCR and Western blot respectively. The proliferation of HASMCs was examined by cell cycle analysis and MTT colorimetric assay. Results After stimulation with PMA,the levels of phosphorylated PΚCα,NF-κB activity and the expression of Cyclin D1 were increased and proliferation of HASMCs enhanced as compared with those of the control group (P<0.05,n=4). After HASMCs were transfected with PΚCα-asODN,the levels of phosphorylated PΚCα were decreased,the NF-κB activity and the expression of Cyclin D1 were significantly reduced and proliferation weakened as compared with those of the PMA treated alone group (P<0.05,n=4). After administration of PDTC,the levels of phosphorylated PΚCα were increased,but the expression of Cyclin D1 and proliferation of HASMCs were significantly reduced as compared with those of the PMA treated alone group (P<0.05,n=4). Conclusion NF-κB is one of downstream regulators of PΚCα. PΚCα-NF-κB cascade is involved in the PMA-induced up-regulation of Cyclin D1 and proliferation in atopic asthmatic sensitized HASMCs.更多还原