CAJ | 학술논문

目的探讨miR-149在SiO2诱导的肺纤维化中对白细胞介素-6(IL-6)表达水平的调节作用.方法 (1)建立SiO2粉尘诱导的小鼠肺纤维化模型,实时荧光定量聚合酶链式反应(quantitative real-time polymerase chainreaction,qRT-PCR)检测小鼠肺组织miR-149表达,蛋白免疫印迹法(Western blot)和免疫组织化学法观察白细胞介素-6(IL-6)的蛋白表达情况.(2)应用具有肺泡Ⅱ型上皮细胞特性的(A549)及支气管上皮细胞(HBE)构建SiO2粉尘刺激的呼吸系统上皮细胞模型,应用qRT-PCR检测miR-149水平,用Western blot法检测IL-6蛋白表达情况.(3)通过体外转染miR-149相似剂(mimics)及抑制剂(inhibitors)至A549细胞,Westem blot法检测细胞表达炎症因子IL-6蛋白的表达情况.(4)双抗夹心酶联免疫吸附试验(ELISA)检测煤工尘肺患者血清中IL-6蛋白的表达情况.结果 (1)在SiO2诱导的小鼠肺纤维化模型中,与空白对照组比较,SiO2处理后的3个时间点,小鼠肺组织miR-149表达明显下调,而IL-6蛋白表达明显上调,差异有统计学意义(P＜0.01).(2)在粉尘刺激上皮细胞模型中,A549细胞及HBE细胞经SiO2刺激后,IL-6蛋白表达上调,miR-149表达量明显下调,差异有统计学意义(P＜0.01).(3)对A549细胞转染miR-149相似物及抑制剂后实验结果显示,高表达miR-149后,A549细胞IL-6蛋白表达下调,反之亦然.(4)与对照组比较,Ⅱ期、Ⅲ期煤工尘肺组血清IL-6水平明显升高,差异有统计学意义(P＜0.01).结论 (1)SiO2粉尘所诱导的肺纤维过程中miR-149表达降低,IL-6蛋白表达增高.(2)miR-149可负调控细胞IL-6的表达水平.
목적 탐토miR-149재SiO2유도적폐섬유화중대백세포개소-6(IL-6)표체수평적조절작용.방법 (1)건립SiO2분진유도적소서폐섬유화모형,실시형광정량취합매련식반응(quantitative real-time polymerase chainreaction,qRT-PCR)검측소서폐조직miR-149표체,단백면역인적법(Western blot)화면역조직화학법관찰백세포개소-6(IL-6)적단백표체정황.(2)응용구유폐포Ⅱ형상피세포특성 적(A549)급지기관상피세포(HBE)구건SiO2분진자격적호흡계통상피세포모형,응용qRT-PCR검측miR-149수평,용Western blot법검측IL-6단백표체정황.(3)통과체외전염miR-149상사제(mimics)급억제제(inhibitors)지A549세포,Westem blot법검측세포표체염증인자IL-6단백적표체정황.(4)쌍항협심매련면역흡부시험(ELISA)검측매공진폐환자혈청중IL-6단백적표체정황.결과 (1)재SiO2유도적소서폐섬유화모형중,여공백대조조비교,SiO2처리후적3개시간점,소서폐조직miR-149표체명현하조,이IL-6단백표체명현상조,차이유통계학의의(P＜0.01).(2)재분진자격상피세포모형중,A549세포급HBE세포경SiO2자격후,IL-6단백표체상조,miR-149표체량명현하조,차이유통계학의의(P＜0.01).(3)대A549세포전염miR-149상사물급억제제후실험결과현시,고표체miR-149후,A549세포IL-6단백표체하조,반지역연.(4)여대조조비교,Ⅱ기、Ⅲ기매공진폐조혈청IL-6수평명현승고,차이유통계학의의(P＜0.01).결론 (1)SiO2분진소유도적폐섬유과정중miR-149표체강저,IL-6단백표체증고.(2)miR-149가부조공세포IL-6적표체수평.
Objective To investigate the regulatory effect of miR-149 on interleukin-6 (IL-6) expression in silica-induced pulmonary fibrosis.Methods A mouse model of pulmonary fibrosis was established using silica dust; the level of miR-149 in the lung tissues of mice with silica-induced pulmonary fibrosis was measured by quantitative real-time polymerase chain reaction (qRT-PCR),while the protein expression of IL-6 was measured by immunohistochemistry and Western blot.Type Ⅱ alveolar epithelial cells (A549) and bronchial epithelial cells (HBE) were exposed to silica dust to establish a model; the level of miR-149 was measured by qRT-PCR,while the protein expression of IL-6 was measured by Western blot.A549 cells were transfected with miR-149 mimics and inhibitor in vitro,and the cellular expression of IL-6 was measured by Western blot.Serum samples from patients with coal workers' pneumoconiosis were examined by double-antibody sandwich ELISA to measure the protein expression of IL-6.Results At three time points after silica treatment,the miR-149 expression in lung tissues was significantly down-regulated while an evident increase in IL-6 expression was observed in lung tissues (P＜0.01).Silica-stimulated epithelial cells (A549 and HBE) had up-regulated IL-6 expression and down-regulated miR-149 expression (P＜0.01).Increased levels of miR-149 attenuated IL-6 expression,whereas adverse results were found when miR-149 was inhibited.Compared with that in control group,serum level of IL-6 was significantly increased in patients with stage Ⅱ and Ⅲ coal workers' pneumoconiosis (P＜0.01).Conclusion Down-regulation of miR-149 and up-regulation of IL-6 might be involved in the progression of silica-induced pulmonary fibrosis; miR-149 could negatively regulate IL-6 expression.