Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
Chin Med J (Engl). 2010 Jul;123(13):1709-14.
Increased proliferation of airway smooth muscle cells (ASMCs) are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract (CSE) on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle.
ASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 (ascyclin D1) group, CSE group, CSE + pcDNA3.1 group and CSE + pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR (RT-PCR) and Western blotting.
(1) The percentage of S + G2M phase, absorbance value at 490 nm wavelength (A(490)) and the expression rate of PCNA protein in CSE group were (31.22 +/- 1.17)%, 0.782 +/- 0.221, (90.2 +/- 7.0)% respectively, which were significantly increased compared with those of control group ((18.36 +/- 1.02)%, 0.521 +/- 0.109, and (54.1 +/- 3.5)%, respectively) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S + G2M phase, A(490) and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells (P < 0.01). (2) The ratios of A(490) of cyclin D1 mRNA in CSE group was 0.288 +/- 0.034, which was significantly increased compared with that of control group (0.158 +/- 0.006) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 mRNA in ASMCs was much lower than in untreated cells (P < 0.01). (3) The ratios of A(490) of cyclin D1 protein expression in CSE group was 0.375 +/- 0.008, which was significantly increased compared with that of control group (0.268 +/- 0.004) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 protein expression in ASMCs was much lower than in untreated cells (P < 0.01).
CSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.
在哮喘患者中观察到气道平滑肌细胞（ASMC）的增殖增加，吸烟可以加速哮喘中 ASMC 的增殖。为了阐明导致这些变化的分子机制，我们在体外研究了香烟烟雾提取物（CSE）对 ASMC 增殖和细胞周期中重要调节蛋白细胞周期蛋白 D1 表达的影响。
研究了从 8 只棕色挪威大鼠培养的 ASMC。研究中使用了第 3 至 6 代之间的细胞，并根据干预条件将其分为对照组、pcDNA3.1 组、pcDNA3.1-反义细胞周期蛋白 D1（反义 cyclin D1）组、CSE 组、CSE + pcDNA3.1 组和 CSE + pcDNA3.1-反义 cyclin D1 组。通过细胞周期分析、MTT 比色法和增殖细胞核抗原（PCNA）免疫细胞化学染色检测 ASMC 的增殖。通过逆转录-聚合酶链反应（RT-PCR）和 Western 印迹检测 cyclin D1 的表达。
（1）CSE 组 S+G2M 期的百分比、490nm 波长的吸光度值（A（490））和 PCNA 蛋白的表达率分别为（31.22±1.17）%、0.782±0.221 和（90.2±7.0）%，明显高于对照组（18.36±1.02）%、0.521±0.109 和（54.1±3.5）%）（P<0.01）。反义 cyclin D1 质粒转染 30 小时后，ASMC 的 S+G2M 期百分比、A（490）和 PCNA 蛋白的表达率明显低于未处理细胞（P<0.01）。（2）CSE 组 cyclin D1mRNA 的 A（490）比值为 0.288±0.034，明显高于对照组的 0.158±0.006（P<0.01）。反义 cyclin D1 质粒转染 30 小时后，ASMC 中 cyclin D1mRNA 的 A（490）比值明显低于未处理细胞（P<0.01）。（3）CSE 组 cyclin D1 蛋白表达的 A（490）比值为 0.375±0.008，明显高于对照组的 0.268±0.004（P<0.01）。反义 cyclin D1 质粒转染 30 小时后，ASMC 中 cyclin D1 蛋白表达的 A（490）比值明显低于未处理细胞（P<0.01）。
CSE 可能通过调节 cyclin D1 表达来增加哮喘大鼠 ASMC 的增殖。
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