siRNA沉默YKL-40对支气管哮喘小鼠气道平滑肌增殖及迁移的影响

Effect of silencing YKL-40 by siRNA on proliferation and migration of airway smooth muscle cells in asthmatic mice

摘要:

目的探讨小干扰核糖核酸（siRNA）沉默人软骨糖蛋白39（YKL-40）对支气管哮喘小鼠气道平滑肌增殖及迁移的影响。方法20 只健康雌性BALB/c 小鼠随机分为健康组（ n=10）和哮喘组（n =10），采取腹腔注射抗原和雾化吸入卵蛋的方式复制哮喘模型，健康组小鼠处理与哮喘组相同，只是将致敏物换成生理盐水，留取气管和支气管进行细胞培养，两组小鼠气道壁平滑肌细胞根据转染物不同分为siRNA-YKL-40 组、阴性对照组和空白对照组，MTT法检测不同转染组小鼠气道壁平滑肌细胞增殖情况，Transwell 法检测不同转染组 小鼠气道壁平滑肌细胞迁移能力，实时荧光定量聚合酶链反应（qRT-PCR）和蛋白免疫印迹法（Western blot）检测不同转染组小鼠气道壁平滑肌细胞中YKL-40、白细胞介素4（IL-4）和干扰素-γ（IFN-γ）基因和蛋白表达。结果哮喘组中siRNA-YKL-40组48～96h时细胞吸光度A值均低于阴性对照组和空白对照组，哮喘组中siRNA-YKL-40 组、阴性对照组和空白对照组48～96 h 时细胞吸光度A 值均高于健康组，差异有统计学意义（p <0.05）；哮喘组中siRNA-YKL-40 组迁移细胞数（86.38±8.61）个，低于阴性对照组和空白对照组，哮喘组迁移细胞数均高于健康组，差异有统计学意义（p<0.05）；哮喘组和健康组中siRNA-YKL-40 组-40 基因和蛋白相对表达量均低于阴性对照组和空白对照组（ p<0.05），哮喘组中siRNA-YKL-40 组-4 基因和蛋白相对表达量均低于阴性对照组和空白对照组，而-γ基因和蛋白相对表达量与IFN-γ/IL-4均高于阴性对照组和空白对照组（p <0.05），与健康组比较，哮喘组中siRNA-YKL-40 组、阴性对照组和空白对照组-4 基因和蛋白相对表达量均升高，而- 基因和蛋白相对表达量和IFN-γ/IL-4均降低，差异有统计学意义（p <0.05）。结论靶向-40 基因沉默能抑制气道壁平滑肌细胞增殖及迁移，可能与调节IL-4/IFN-γ失衡状态而减少气道炎症反应有关。

Abstract:

Objective To investigate the effects of silencing human cartilage glycoprotein-39 ( YKL-40 ) by small interfering ribonucleic acid (siRNA) on the proliferation and migration of airway smooth muscle cells in asthmatic mice. Methods Twenty healthy female BALB/c mice were randomly divided into healthy group (n =10) and asthma group (n = 10). Asthma models were established by intraperitoneal injection of antigens and inhalation of ovum (OVA). The mice in the healthy group were treated the same as the asthma group, except that the sensitizers were replaced by saline. Tracheal and bronchial tubes were used for cell culture.According to the different transfectants, the airway smooth muscle cells of the two groups were divided into siRNA-YKL-40 group, negative control group and blank control group. The proliferation of airway smooth muscle cells of mice in different transfected groups were detected by MTT assay. The migration abilities of airway smooth muscle cells of mice in different transfected groups were detected by Transwell assay. The expressions of YKL-40, interleukin 4 (IL-4 ) and interferon-γ( IFN-γ ) genes and proteins in airway smooth muscle cells in different transfected groups were detected by qRT-PCR and Western blot, respectively. Results In the asthma group, the absorbance A values at 48, 72 and 96 h of the siRNA-YKL -40 group were significantly lower than the negative control group and blank control group and the absorbance A values at 48, 72 and 96 h of the siRNA-YKL-40 group, negative control group and blank control group in the asthma group were significantly higher than the healthy group, (p < 0.05). In the asthma group, the number of migrated cells of the siRNA-YKL-40 group was (86.38 ±8.61), which was significantly lower than the negative control group and blank control group and the number of migrating cells of the asthma group was significantly higher than the healthy group (p < 0.05). In the asthma group and the healthy group, the relative expression levels of YKL-40gene and protein in the siRNA-YKL-40 group were lower than the negative control group and blank control group ( p< 0.05), in the asthma group, the relative expression levels of IFN-γ gene and protein in the siRNA-YKL-40 group were lower than the negative control group and blank control group, while the relative expression levels of gene and protein and the value of IFN-γ/IL-4 were significantly higher than the negative control group and blank control group (p < 0.05). Compared with the healthy group, the relative expression levels of - gene and protein in the siRNA-YKL-40 group, negative control group and blank control group in the asthmatic group were significantly increased, while the relative expression levels of IFN-γ gene and protein and the value of IFN-γ/IL-4 were significantly decreased (P <0.05). Conclusions Gene silencing targeting YKL -40 inhibits airway smooth muscle cell proliferation and migration. It might be related to the regulation of the imbalance of IL-4/IFN-γto reduce airway inflammation.