香烟提取物对气道平滑肌细胞增殖作用的研究

Effects of tobacco extract on proliferation of human airway smooth muscle cells

目的探讨香烟提取物(CSE)对人气道平滑肌细胞(ASMCs)增殖以及钙网织蛋白(CRT)、CCAAT增强子结合蛋白α(CEBPα)表达的影响及机制。方法 (1)通过收集经不同浓度CSE处理24 h的ASMCs,分为对照组、2.5%CSE组、5%CSE组、10%CSE组。以MTT法分析各组细胞增殖情况,采用逆转录聚合酶链反应(RT-PCR)检测各组细胞CEBPα的m RNA水平;免疫印迹法检测4组细胞CRT、CEBPα的蛋白水平。(2)在10%CSE组,分别在ASMCs中转染阴性对照si RNA、CRT的si RNA,比较2组ASMCs的CEBPα、CRT表达及细胞增殖情况。结果 (1)对照组、2.5%CSE组、5%CSE组、10%CSE组ASMCs的CEBPα蛋白表达依次递减,CRT和细胞增殖程度呈依次增高(P<0.05);而CEBPαm RNA表达差异无统计学意义(P>0.05)。(2)在10%CSE作用下,CRTsi RNA组中ASMCs的CEBPα表达较阴性对照si RNA组增强(P<0.05),而CRT和细胞增殖程度均较相应阴性对照si RNA组减弱(P<0.05)。结论 CSE可使ASMCs中CRT的表达增强,从而抑制CEBPαm RNA的翻译,使CEBPα的表达减少,最终促进ASMCs的增殖。

Objective To explore the effects and mechanism of cigarette smoke extract （CSE） on the proliferation of airway smooth muscle cells （ASMCs） and the expression of CCAAT/enhancer-binding protein （CEBPα） and calreticulin. Methods （1） The ASMCs were stimulated with different concentrations of CSE for twenty-four hours. According to the concentrations of CSE,the cells were divided into control group, 2.5%CSE group, 5%CSE group and 10%CSE group. The proliferation of ASMCs was measured by MTT colrimetric method. The CEBPαmRNA was analyzed by RT-PCR. Western bloting assay was performed to detect the levels of CRT and CEBPαprotein. （2） In 10%CSE group, transfection of the siRNA respectively for negative control or calreticulin was performed in accordance with instructions. The cell proliferation and the expression of calreticulin and CEBPαwere compared in negative control siRNA group and calreticulin siRNA group. Results （1） With the increasing of the concentrations of CSE, the protein expression of CEBPαdecreased gradually （P〈0.05）, while the proliferation of ASMCs and the protein expression of calreticulin increased （P〈0.05）, but the expression of CEBPαmRNA in ASMCs showed no significant difference in groups with different concentrations of CSE （P〉0.05）. （2） Under the 10%CSE, the expression of CEBPαwas significantly higher in CRT siRNA group than that in negative control group （P〈0.05）,but the cell proliferation and CRT were significantly lower in the calreticulin siRNA group than those in negative control siRNA group （P〈0.05）. Conclusion The CSE exposure contributes to the expression of calreticulin protein,and then inhibits the translation of CEBPαmRNA,thus promotes the proliferation of ASMCs.