整合素β1基因沉默对支气管哮喘小鼠气道平滑肌功能的影响

Effect of gene silencing of integrin β1 on the proliferation and secretory function of airway smooth muscle in asthmatic mice

目的 探讨整合素β1基因沉默对支气管哮喘（简称哮喘）小鼠气道平滑肌细胞（ASMC）增殖和分泌功能的影响.方法 原代培养正常及哮喘模型小鼠ASMC,分别取各自第5代ASMC进行实验,利用RNA干扰（RNAi）技术沉默ASMC的整合素β1基因,RT-PCR技术观察沉默前后ASMC整合素β1 mRNA表达水平变化,蛋白印迹检测沉默前后ASMC整合素β1蛋白含量变化;四甲基偶氮唑盐（MTT）法检测整合素β1基因沉默前后ASMC细胞增殖变化;流式细胞仪测定整合素β1基因沉默前后ASMC细胞周期分布及细胞凋亡率变化情况;ELISA检测整合素β1 基因沉默前后ASMC分泌的白细胞介素（IL）-6及受激活调节正常T细胞表达和分泌因子（RANTES）的含量变化.结果 （1）哮喘PBS对照组RT-PCR显示整合素β1 mRNA（0.907±0.041）较正常PBS对照组（0.527±0.027）显著增高（t=24.632,P＜0.05）,哮喘siRNA干预组（0.503±0.034）较哮喘PBS对照组明显减低（t=24.079,P＜0.05）,正常siRNA干预组较正常PBS对照组无显著性变化（t=1.077,P＞0.05）;（2）Western blot检测显示哮喘PBS对照组整合素β1蛋白表达（0.733±0.067）较正常PBS对照组（0.386±0.044）显著增高（t=13.622,P＜0.05）,哮喘siRNA干预组（0.453±0.074）较哮喘PBS对照组显著减低（t=8.880,P＜0.05）,正常siRNA干预组较正常PBS对照组无显著性变化（t=1.908,P＞0.05）;（3）MTT实验显示3～5 d的吸光度（A）值哮喘PBS对照组较同期正常PBS对照组 显著增强（均P＜0.05）,哮喘siRNA干预组的A值较同期哮喘PBS对照组显著降低（均P＜0.05）,正常siRNA干预组与正常PBS对照组差异无统计学意义（均P＞0.05）;（4）哮喘PBS对照组ASMC处于增殖状态的S期＋G2/M期ASMC比例为[（49±4）%],明显高于正常PBS对照组[（34±4）%]（t=8.035,P＜0.05）,哮喘siRNA干预组ASMC的S期＋G2/M期ASMC比例（42±7）%明显低于哮喘PBS对照组（t=2.212,P＜0.05）,正常siRNA干预组与正常PBS对照组差异无统计学意义（t=0.699,P＞0.05）;（5）哮喘PBS对照组的凋亡率[（3.9±1.4）%]较正常PBS对照组[（7.4±0.5）%]显著升高（t=7.465,P＜0.05）,哮喘siRNA干预组凋亡率[（12.6±2.4）%]明显高于哮喘PBS对照组（t=9.839,P＜0.05）,正常siRNA干预组凋亡率与正常PBS对照组相比差异无统计学意义（t=2.094,P＞0.05）;（6）哮喘PBS对照组IL-6[（545±28）ng/L]、RANTES分泌值[（345±28）ng/L]高于正常PBS对照组[分别为（219±26）ng/L和（138±16）ng/L,均P＜0.05],哮喘siRNA干预组分泌量[分别为（348±26）ng/L和（250±24）ng/L]显著低于哮喘PBS对照组（t值分别为6.192和4.590,均P＜0.05）,而正常siRNA干预组较正常PBS对照组差异无统计学意义（均P＞0.05）.结论 靶向ASMC整合素β1基因沉默能抑制哮喘模型小鼠ASMC的增殖、降低分泌功能并促进ASMC凋亡.

Objectiye To investigate the effect of gene silencing of integrin β1 on the proliferation and secretory function of airway smooth muscle cells （ASMC） in asthmatic mice.Methods Integrin β1 gene was silenced by using RNAi technology in the fifth generation ASMC of normal and asthmatic mice.The integrin β1 mRNA expression and integrin β1 protein were analyzed by RT-PCR and Western blot respectively before and after the integrin β1 gene silencing in ASMC.The cell proliferation changes of ASMC were measured by MTT assay before and after gene silencing of integrin β1.The cell cycle distribution and apoptosis rate were analyzed by flow cytometry.The expression of interleukin （ IL）-6 and RANTES were analyzed by ELISA assay.Results （ 1 ） Integrin β1 mRNA expression by RT-PCR （0.907 ± 0.041 ） was significantly higher in asthma control group compared with normal control group （0.527 ± 0.027 ） （ t =24.632,P 〈0.05）,and was significantly decreased in asthma siRNA intervention group （0.503 ±0.034）compared with asthma control group （ t = 24.079,P 〈 0.05 ）.There was no significant difference in the normal group compared with the intervention group （ t = 1.077,P 〉 0.05 ）.（ 2 ） Integrin β1 protein expression （0.733 ± 0.067 ） was significantly higher（ t = 13.622,P 〈 0.05 ） in the asthma control group compared with normal control group（0.386 ± 0.044）,and was significantly reduced in the asthma siRNA intervention group （0.453 ± 0.074） compared with the control group （t = 8.880 ,P 〈 0.05 ）.There was no significant change in the normal control group after the intervention （ t = 1.908,P 〉 0.05 ）.（ 3 ） The absorbance value was significantly enhanced in the asthma control group compared with the same period of normal control group （ t = 9.528,5.799,3.372,all P 〈 0.05 ）,and was significantly reduced in asthma siRNA intervention group compared with the same period of asthma control group （t =2.684,2.546,2.897,all P 〈0.05） ,and no significant changes in normal group after the intervention（t =0.067,1.198,0.589 ,all P 〉 0.05 ）.（4） The S ＋ G2/M phase ratio of ASMC was significantly higher in asthma control group[（49 ±4） %] compared with normal control group （34 ±4）% （t = 8.035,P 〈 0.05 ）,and was significantly lower in the asthma siRNA intervention group [（42 ±7）%] compared with asthma control group （t =2.212,P〈0.05 ）,and no significant changes in normal control group after the intervention （ t = 0.699,P 〉 0.05 ）.（5）The apoptosis rate was significantly lower in asthma control group [（3.9 ± 1.4） %] compared with normal control group [（7.4 ± 0.5 ） %] （ t = 7.465,P 〈 0.05 ）,and was significantly higher in asthma siRNA intervention group[（ 12.6 ± 2.4） %] compared with asthma control group （ t = 9.839,P 〈 0.05 ）,but no significant changes in normal control group after the intervention（ t =2.094 ,P 〉0.05 ）.（6） The secreted IL6 [（545 ±28）ng/L] and RANTES [（345 ± 28）ng/L] were higher in asthma control group compared with the normal control group [（219 ± 26 ） ng/L,（ 138 ± 16 ） ng/L,respectively,t = 26.789,20.451,all P 〈0.05],and was significantly decreased in the asthma siRNA intervention group [（347 ± 26）ng/L,（250 ±24） ng/L] compared with asthma control group（ t = 6.192,4.590,all P 〈 0.05 ）,while there was no significant difference in the normal control group after the intervention.Conclusion Gene silencing targeting integrin β1 inhibited proliferation and secretion,but promoted apoptosis of ASMC from asthmatic mice.