天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (10): 1137-1140.doi: 10.11958/j.issn.0253-9896.2015.10.014

• 实验研究 • 上一篇    下一篇

microRNA-21对肺成纤维细胞增殖和分化的影响

  

  1. 南华大学附属南华医院儿科 （邮编 421001）
  • 收稿日期:2014-12-10 修回日期:2015-05-18 出版日期:2015-10-15 发布日期:2015-10-22

Effect of microRNA-21 on proliferation and differentiation of pulmonary fibroblasts of mice

  1. Pediatrics of the Affiliated Nanhua Hosital of University of South China， Hengyang 421001， China
  • Received:2014-12-10 Revised:2015-05-18 Published:2015-10-15 Online:2015-10-22

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摘要：

摘要： 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 50 μL Opti-MEM 加入 2.5 μL
PBS、 阴性对照储存液和 miR-21 mimic 储存液（20 μmol/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。

关键词: 肺纤维化, 成纤维细胞, 转化生长因子β1, 微 RNAs, 细胞增殖, 细胞分化, ADAMTS-1, microRNA-21

Abstract: Abstract： 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 (2.5 μL), negative control stock solution and miR-21 mimic stock solution (20 μmol/L) were
added into Opti-MEM (50 μL) 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.

Key words: pulmonary fibrosis, fibroblasts, transforming growth factor beta1, microRNAs, cell proliferation, cell differ?
entiation, ADAMTS-1, microRNA-21