核因子κB在被动致敏的人气道平滑肌细胞增殖中的信号转导作用

目的探讨核因子κB(NF-κB)是否参与被动致敏的人气道平滑肌细胞(HASMC)增殖及是否是蛋白激酶C(PKC)激活后的下游途径.方法用10%哮喘患者血清被动致敏HASMC,以10%非哮喘者血清为对照,并用吡咯烷二硫氨基甲酸(PDTC)及12-肉豆蔻酰-13-乙酸佛波酯(PMA)干预HASMC,用流式细胞术、MTT法及增殖细胞核抗原(PCNA)免疫荧光技术检测HASMC增殖;NF-κBp65免疫荧光技术及电泳迁移率改变分析(EMSA)检测NF-κB活性.结果 (1)哮喘血清处理的HASMC,S期细胞比例、吸光度值(A值)、PCNA表达阳性率、NF-κBp65阳性率及EMSA灰度值均较对照血清组增加(均P<0.05),PDTC预处理后上述指标均下降 (均P<0.05).(2)PMA+哮喘血清处理HASMC后,S期细胞比例、A值、PCNA表达阳性率、NF-κBp65阳性率及EMSA灰度值分别为(25.52±3.38)%、0.572±0.054、 (81.2±10.2)%、(26.5±5.0)%和71 654±12 293,PDTC预处理后上述指标均下降(均P<0.05).结论 NF-κB参与了哮喘血清被动致敏的HASMC增殖,在其增殖中存在着PKC/NF-κB信号途径.

Effect of nuclear factor-kappaB on signal transduction of passively sensitized human airway smooth muscle cells proliferation

OBJECTIVE: To investigate whether nuclear factor-kappaB (NF-kappaB) contributes to passively sensitized human airway smooth muscle cell (HASMCs) proliferation, and whether it is the downstream factor of activated protein kinase C (PKC). METHODS: HASMCs in culture were passively sensitized with 10% serum from asthmatic patients, with non-asthmatic human serum treated HASMCs as the control. NF-kappaB specific inhibitor pyrrolidine dithiocarbamate (PDTC) and PKC agonist phorbol 12-myristate 13-acetate (PMA) were used to intervene HASMCs exposed to asthmatic serum and non-asthmatic control serum. The proliferation of HASMCs was examined by cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining respectively. NF-kappaB activity was detected by NF-kappaBp65 immunofluorescence staining and electrophoretic mobility shift assay (EMSA) respectively. RESULTS: (1) The percentage of S phase, A value, the positive expression rate of PCNA, the positive expression rate of NF-kappaBp65 and EMSA value in HASMCs passively sensitized with asthmatic serum were (21.78 +/- 2.79)%, 0.466 +/- 0.058, (67.5 +/- 8.5)%, (12.6 +/- 2.2)% and 32 781 +/- 9499 respectively. They were significantly increased compared with those of the control serum group (P < 0.05). After previously treated with PDTC, the above figures were decreased to (16.37 +/- 3.05)%, 0.389 +/- 0.035, (53.4 +/- 5.1)%, (4.9 +/- 1.3)% and 3934 +/- 937 respectively (P < 0.05). (2) After HASMCs were treated with both PMA and asthmatic serum, the percentage of S phase, A value, the positive expression rate of PCNA, the positive expression rate of NF-kappaBp65 and EMSA value were (25.52 +/- 3.38)%, 0.572 +/- 0.054, (81.2 +/- 10.2)%, (26.5 +/- 5.0)% and 71 654 +/- 12 293 respectively. After previously treated with PDTC, the above figures were (16.42 +/- 2.72)%, 0.386 +/- 0.031, (54.2 +/- 5.3)%, (5.9 +/- 1.4)% and 4808 +/- 1084 respectively. The difference was significant (P < 0.05). CONCLUSIONS: NF-kappaB may contribute to the proliferation of HASMCs passively sensitized with human asthmatic serum, which involves the PKC/NF-kappaB signal pathway.