CAJ | 학술논문

目的：探讨小鼠肺成纤维化细胞模型中microRNA（miR）-21对肺成纤维细胞增殖和分化的影响。方法24只SPF级C57BL/6小鼠随机分为假手术组和肺纤维化模型组，各12只。经气管内注入博莱霉素建立小鼠肺纤维化模型，采用荧光定量PCR检测各组肺组织miR-21的表达。提取小鼠成纤维细胞，胰酶消化，接种于6孔板，使细胞浓度达到30%~50%。设随机对照组、空白对照组和miR-21 mimic组。各组分别在50μL Opti-MEM加入2.5μL PBS、阴性对照储存液和miR-21 mimic储存液（20μmol/L）。采用细胞计数试剂盒-8(CCK-8)法检测细胞增殖活性，蛋白质印迹法检测成纤维细胞含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS-1)和转化生长因子（TGF）-β1的表达。结果与假手术组比较，肺纤维化模型组小鼠肺组织中miR-21的表达量明显上调。与随机对照组和空白对照组比较，miR-21 mimic组成纤维细胞miR-21基因表达显著上调，成纤维细胞增殖率增加，ADAMTS-1蛋白表达明显下降，而TGF-β1表达显著上调；空白对照组与随机对照组ADAMTS-1及TGF-β1蛋白表达无明显差异。结论在小鼠肺成纤维化细胞模型中上调的miR-21可通过ADAMTS-1/TGF-β1信号通路促进肺纤维化。
목적：탐토소서폐성섬유화세포모형중microRNA（miR）-21대폐성섬유세포증식화분화적영향。방법24지SPF급C57BL/6소서수궤분위가수술조화폐섬유화모형조，각12지。경기관내주입박래매소건립소서폐섬유화모형，채용형광정량PCR검측각조폐조직miR-21적표체。제취소서성섬유세포，이매소화，접충우6공판，사세포농도체도30%~50%。설수궤대조조、공백대조조화miR-21 mimic조。각조분별재50μL Opti-MEM가입2.5μL PBS、음성대조저존액화miR-21 mimic저존액（20μmol/L）。채용세포계수시제합-8(CCK-8)법검측세포증식활성，단백질인적법검측성섬유세포함Ⅰ형혈소판결합단백기서적해취단백양금속단백매(ADAMTS-1)화전화생장인자（TGF）-β1적표체。결과여가수술조비교，폐섬유화모형조소서폐조직중miR-21적표체량명현상조。여수궤대조조화공백대조조비교，miR-21 mimic조성섬유세포miR-21기인표체현저상조，성섬유세포증식솔증가，ADAMTS-1단백표체명현하강，이TGF-β1표체현저상조；공백대조조여수궤대조조ADAMTS-1급TGF-β1단백표체무명현차이。결론재소서폐성섬유화세포모형중상조적miR-21가통과ADAMTS-1/TGF-β1신호통로촉진폐섬유화。
Objective To investigate the effect of microRNA(miR)-21 on proliferation and differentiation of murine pulmonary fibroblasts. Methods C57BL/6 mice of SPF grade (n=24) were randomly divided into Sham group and Pulmo?nary fibrosis model group with 12 mice in each group. Pulmonary fibrosis model was established by trans-tracheal jet ventila?tion of bleomycin into mice. The transcription levels of miR-21 were examined by quantitative real-time PCR in various pul?monary fibrosis tissues. Primary fibroblast were isolated and digested by Trypsin then inoculated into 6 well plate to reach confluence of 30%-50%. PBS (2.5μL), negative control stock solution and miR-21 mimic stock solution (20μmol/L) were added into Opti-MEM (50μL) as control group, blank group and miR-21 mimic group respectively.The cell viability was as?sessed by CCK-8. Expressions of ADAMTS-1 and TGF-β1 in the pulmonary fibroblasts were tested using Western blot. Re?sults The expression of miR-21 was significantly increased in lungs of mice in pulmonary fibrosis model group than that in sham group. Expression of miR-21 was higher in miR-21 mimic group than that in control group and blank group. Expres?sion of miR-21 was significantly higher with better cell viability in miR-21 mimic group than that in control group and blank group. The expression of ADAMTS-1 was significantly decreased in miR-21mimic group, while the expression of TGF-β1, a target gene of miR-21, was significantly increased in miR-21 mimic group compared with the other two groups. There is no significant different in expressions of ADAMTS-1 and TGF-β1 between control group and blank group. Conclusion Over?expression of miR-21 in pulmonary fibroblasts disrupts TGF-β1 signaling pathway by reducing expression of ADAMTS-1, which promotes the proliferation and differentiation of pulmonary fibroblast.