Xu Shu-yun, Xu Yong-jian, Zhang Zhen-xiang, Ni Wang, Chen Shi-xin
Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Chin Med J (Engl). 2004 Jan;117(1):30-6.
Airway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation.
HASMCs in culture were passively sensitized with 10% serum from asthmatic patients, with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-alpha in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction.
The percentage of S phase, absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30 +/- 2.68)%, 0.430 +/- 0.060 and (63.4 +/- 7.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [(10.01 +/- 1.38)%, 0.328 +/- 0.034 and (37.2 +/- 4.8)%, respectively] (P < 0.05). After HASMCs were passively sensitized with asthmatic serum, they were treated with PMA, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (20.33 +/- 3.39)%, 0.542 +/- 0.065 and (76.0 +/- 8.7)% respectively, which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA(P < 0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (11.21 +/- 1.56)%, 0.331 +/- 0.047 and (38.8 +/- 6.0)% respectively, which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 (P < 0.05). The relative ratio of value A of PKC-alpha mRNA and the positive percentage of PKC-alpha protein expression in passively sensitized HASMCs were 1.23 +/- 0.10 and (61.1 +/- 9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05 +/- 0.09 and (34.9 +/- 6.7)%, respectively] (P < 0.05).
The proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.
气道平滑肌增殖在哮喘气道重塑中起重要作用。但关于哮喘中气道平滑肌细胞增殖的细胞内信号通路知之甚少。本文旨在研究蛋白激酶C（PKC）及其α亚型对被动致敏的人气道平滑肌细胞（HASMCs）增殖的作用。
用哮喘患者的10%血清对培养的HASMCs进行被动致敏，以非哮喘患者血清处理的HASMCs作为对照。通过细胞周期分析、3-（4,5-二甲基-2-噻唑基）-2,5-二苯基四氮唑溴盐（MTT）比色法和增殖细胞核抗原（PCNA）免疫荧光染色检测HASMCs的增殖情况。还用相同方法检测了PKC激动剂佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）和PKC抑制剂Ro-31-8220对暴露于哮喘患者血清和非哮喘对照血清的HASMCs增殖的影响。通过免疫荧光染色和逆转录-聚合酶链反应检测被动致敏的HASMCs中PKC-α的蛋白和mRNA表达。
用哮喘患者血清被动致敏的HASMCs中S期百分比、吸光度（A值）和PCNA蛋白表达阳性百分比分别为（16.30±2.68）%、0.430±0.060和（63.4±7.4）%，与用对照血清处理的HASMCs相比显著增加[分别为（10.01±1.38）%、0.328±0.034和（37.2±4.8）%]（P<0.05）。用哮喘患者血清对HASMCs进行被动致敏后，再用PMA处理，S期百分比、A值和PCNA蛋白表达阳性百分比分别为（20.33±3.39）%、0.542±0.065和（76.0±8.7）%，与未用PMA处理的哮喘患者血清致敏的HASMCs相比显著增加（P<0.05）。用哮喘患者血清被动致敏的HASMCs用Ro-31-8220处理后，S期百分比、A值和PCNA蛋白表达阳性百分比分别为（11.21±1.56）%、0.331±0.047和（38.8±6.0）%，与未用Ro-31-8220处理的哮喘患者血清致敏的HASMCs相比显著降低（P<0.05）。被动致敏的HASMCs中PKC-α mRNA的A值相对比值和PKC-α蛋白表达阳性百分比分别为1.23±0.10和（61.1±9.4）%，与用对照血清处理的HASMCs相比显著增加[分别为1.05±0.09和（34.9±6.7）%]（P<0.05）。
用哮喘患者血清被动致敏的HASMCs增殖增加。PKC及其α亚型可能促成了这种增殖。
