摘要目的 探讨香烟烟雾提取物(CSE)对支气管哮喘(简称哮喘)患者血清致敏的人气道平滑肌细胞(HASMC)增殖作用及其可能机制.方法 原代培养HASMC,取第4～8代细胞,用10%哮喘患者血清致敏HASMC,分为血清组、血清+CSE组、血清+GW8510(嘧啶基-苯磺酰胺,细胞周期蛋白依赖激酶-4抑制剂)组、血清+CSE+GW8510组,以10%非哮喘患者血清为对照组.用流式细胞术、四甲基偶氮唑盐(MTT)法及3H-TdR掺入法检测HASMC增殖,用逆转录-聚合酶链反应(RT-PCR)和Western blot检测细胞周期蛋白D1(cyclinD1])的表达.结果 血清组HASMC的S+G2 M期比例、吸光度(A)值和细胞DNA合成量[分别为(21.4±1.1)%、0.39±0.12和2669±138]明显高于对照组,差异均有统计学意义(q值分别为6.25、5.61、6.82,均P＜0.01);血清+CSE组HASMC的S+G2M期比例、A值和DNA合成量分别为(33.3±1.3)%、0.61±0.20和3552±303,与血清组比较差异均有统计学意义(q值分别为5.67、6.32、5.56,均P＜0.01);血清+GW8510组HASMC的S+G2M期比例、A值和DNA合成量分别为(14.7±1.4)%、0.30±0.10和1812±109,与血清组比较差异均有统计学意义(q值分别为6.02、5.53、5.79,均P＜0.01).血清+CSE组HASMC cyclinD1 mRNAA值比和蛋白表达A值比分别为0.521±0.102和0.312±0.002,与血清组(分别为0.291±0.112和0.186±0.002)比较,差异均有统计学意义(q值分别为12.02和9.26,均P＜0.01);血清+GW8510组分别为0.223±0.038、0.150±0.002,与血清组比较差异均有统计学意义(q值分别为6.86、5.60,均P＜0.01).结论 CSE可能通过cyclinD参与调控哮喘患者血清致敏的HASMC增殖.

abstractsObjective To study the effect of cigarette smoke extract (CSE) on the proliferation of human airway smooth muscle cells (HASMCs) sensitized by serum from asthmatic patients and the underlying mechanisms.Methods HASMCs were cultured from primary generation.Cells between passage 4 and 8 were used in the study.HASMCs were sensitized by 10% serum from asthmatic patients and were divided into an asthmatic serum group, an asthmatic serum + CSE group, an asthmatic serum + GW8510 (inhibitor of cyclin-dependent kinase-4) group and an asthmatic serum + CSE + GW8510 group.Non-asthmatic human serum treated HASMCs served as the control.The proliferation of HASMCs was examined by cell cycle analysis, MTT colorimetric assay and[3H]thymidine incorporation.The expression of cyclinD1 was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.Results The percentage of S + G2/M phase, the absorbance (A) value and the DNA synthesis value in asthmatic serum group were significantly increased compared with those of the control group ( q = 6.25,5.61,6.82,respectively, all P ＜0.01 ).The percentage of S + G2/M phase, the absorbance (A) value and DNA synthesis value in the asthmatic serum group were (21.4 ± 1.1 ) %, 0.392 ± 0.124 and 2669 ± 138,respectively.Their value in the asthmatic serum + CSE group were (33.3 ± 1.3)%, 0.612 ±0.201 and 3552 ± 303, respectively, which were significantly increased compared with those of the asthmatic serum group( q = 5.67,6.32,5.56, respectively, all P ＜ 0.01 ).Their value in the asthmatic serum + GW8510 group were (14.7 ± 1.4)%, 0.301 ±0.097 and 1812 ± 109, respectively, which were significantly decreased compared with those of the asthmatic serum group ( q = 6.02,5.53,5.79, respectively, all P ＜0.01 ).The ratios of A value of cyclinD1 mRNA and the expression of cyclinD1 protein in the asthmatic serum group were 0.291 ±0.112 and 0.186 ±0.002, respectively.The ratios of A value in the asthmatic serum + CSE group were 0.521 ± 0.102 and 0.312 ± 0.002, respectively, which were significantly increased compared with those of the asthmatic serum group (q = 12.09,9.26, respectively, all P ＜ 0.01 ).The ratios of A value in the asthmatic serum + GW8510 group were 0.223 ±0.038 and 0.150 ±0.002,respectively, which were significantly decreased compared with those of the asthmatic serum group (q =6.86,5.60, respectively, all P ＜ 0.01 ).Conclusions HASMCs sensitized by serum from asthmatic patients showed accelerated proliferation after intervention by CSE, with increased expression of cyclinD1.CSE may increase the proliferation of HASMCs sensitized by serum from asthmatic patients via regulating cyclinD expression.