siRNA沉默凋亡抑制蛋白基因对支气管哮喘小鼠气道平滑肌增殖的影响

Effects of siRNA silencing of inhibitor of apoptosis protein gene on the proliferation of airway smooth muscle in bronchial asthma mice

目的探讨siRNA沉默凋亡抑制蛋白基因对支气管哮喘小鼠气道平滑肌增殖的影响。方法将20只雌性BALB/c小鼠分为实验组和对照组,实验组构建哮喘模型,取气管和支气管进行细胞培养。对实验组和对照组培养的气道壁平滑肌细胞分别进行分组：磷酸盐缓冲液（PBS）对照组（加入PBS）、错义链对照组（转染错义链）和siRNA转染组（转染siRNA Survivin）,检测气道壁平滑肌细胞中Survivin基因、Survivin蛋白和caspase-9蛋白表达,检测细胞上清液中IL-6和人趋化因子（CCL5）水平。结果 RT-PCR结果显示,实验组中siRNA转染组小鼠气道壁平滑肌细胞中Survivin RNA相对表达量均低于PBS对照组和错义链对照组,气道壁平滑肌细胞中Survivin蛋白表达量均低于PBS对照组和错义链对照组,而caspase-9蛋白表达量则高于PBS对照组和错义链对照组,差异均有统计学意义（P〈0.05）;实验组中siRNA转染组气道壁平滑肌细胞合成分泌IL-6和CCL5水平均低于PBS对照组和错义链对照组;2~4d细胞增殖量均低于PBS对照组和错义链对照组;气道壁平滑肌细胞G0/G1期比例高于PBS对照组和错义链对照组,而S/G2期比例则低于PBS对照组和错义链对照组,细胞凋亡率则高于PBS对照组和错义链对照组,差异均有统计学意义（P〈0.05）。结论 siRNA特异性沉默Survivin基因,可加速气道壁平滑肌细胞凋亡,有效抑制细胞异常增殖及分泌功能。

Objective To investigate the effects of siRNA silencing of inhibitor of apoptosis protein gene on the proliferation of airway smooth muscle in bronchial asthma mice. Methods Tweenty female BALB/c mice were randomly divided into experimental group and control group. Mice in the experimental group were constructed asthma models. The trachea＇and bronchi were collect- ed for cell culture. Respectively, the cultured airway smooth muscle cells in the experimental group and the control group were grouped：PBS control group （added to PBS solution）,missense chain control group （transfected with missense chain） and siRNA transfection group （transfected with siRNA Survivin）. The expressions of Survivin mRNA,Survivin protein and caspase-9 protein in airway smooth muscle cells were detected. The levels of IL-6 and CCL5 in cell supernatant were detected. Results RT-PCR re suits showed that,the relative expression levels of Survivin RNA of airway smooth muscle cells in the siRNA transfection group of the experimental group were lower than the PBS control group and missense chain group,the differences were statistically significant （P〈0.05）. Western blot results showed that,the expressions of Survivin proteins in the siRNA transfection group of the ex- perimental group were lower than the PBS control group and missense chain control group, while the caspase-9 protein were higher, In the experimental group, the synthesis and secretion levels of IL-6 and CCL5 of airway smooth muscle cells in the siRNA transfec- tion group were lower than the PBS control group and missense chain control group, the cell proliferations at 2--4 d in the siRNA transfection group were lower than the PBS control group and missense chain control group, the proportion of Go/G1 phase in the siRNA transfection group were higher than the PBS control group and missense chain control group, but the proportion of S/G2 phase were lower than the PBS control group and missense chain control group, cells apoptosis rates of the siRNA transfection group were lower than the PBS control group and missense chain control group, the differences were statistically significant（P〈 0.05）. Conclusion siRNA specific silencing Survivin gene could accelerate the airway smooth muscle cell apoptosis,and inhibit cell abnormal proliferation and secretion.