解剖学报

解剖学报 ›› 2014, Vol. 45 ›› Issue (3): 393-397.doi: 10.3969/j.issn.0529-1356.2014.03.018

• 组织学胚胎学发育生物学 • 上一篇    下一篇

白细胞介素-34/集落刺激因子-1R在转化生长因子-β1诱导A549细胞上皮-间质转化中的表达

郑金旭1* 朱勤1 刘超1 宋萍1 端礼荣2   

  1. 1.江苏大学附属医院呼吸科，江苏 镇江 212001; 2.江苏大学基础医学与医学技术学院，江苏 镇江 212013
  • 收稿日期:2014-02-18 修回日期:2014-03-04 出版日期:2014-06-06 发布日期:2014-06-06
  • 通讯作者: 郑金旭 E-mail:jxuzh135@163.com
  • 基金资助:

    上海市自然基金项目;江苏省“333工程”资助项目

Expression of interleukin -34/ colony stimulating factor -1R in the process of transforming growth factor -β1 inducing  A549 cells transition

ZHENG Jin-xu 1* ZHU Qin1 LIU Chao1 SONG Ping1 DUAN Li-rong 2   

  1. 1.Department of Respiratory Medicine, Affiliated Hospital of Jiangsu University, Jiangsu Zhenjiang 212001，China;2.College of Basic Medicine and Medical Technology，Jiangsu University， Jiangsu Zhenjiang 212013, China
  • Received:2014-02-18 Revised:2014-03-04 Online:2014-06-06 Published:2014-06-06
  • Contact: ZHENG Jin-xu E-mail:jxuzh135@163.com

摘要：

目的 探讨白细胞介素-34/集落刺激因子-1受体（IL-34/CSF-1R）在转化生长因子-β1（TGF-β1）诱导A549细胞发生上皮-间质转化（EMT）中的表达。方法 体外培养A549细胞；CCK 8法检测不同浓度TGF-β1在不同时间点对A549细胞增殖的影响；选用5μg/L TGF-β1刺激A549细胞（0、12、24、48h），提取细胞蛋白和RNA，Western blotting法检测α-平滑肌肌动蛋白（α-SMA）、E-钙黏连蛋白（E-Cad）、基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）、基质金属蛋白酶抑制剂-1（TIMP-1）蛋白的变化；Real-time PCR法检测IL-34、CSF-1R、MMP-2、MMP-9基因表达的变化。 结果 TGF-β1对A549细胞的增殖无明显影响（P>0.05）。TGF-β1刺激A549细胞后，间质细胞标志性蛋白α-SMA表达升高，上皮细胞标志性蛋白E-Cad表达下降，纤维化相关的MMP-2蛋白表达升高，MMP-9蛋白表达先升高后下降，TIMP-1蛋白早期出现短暂性增高后，呈持续降低趋势（P<0.01）。IL-34 mRNA表达逐渐升高，CSF-1R mRNA、MMP-2 mRNA、MMP-9 mRNA表达先升高后下降（P<0.01）。 结论 TGF-β1诱导A549细胞的EMT过程中，存在IL-34/CSF-1R的动态表达。

关键词: 转化生长因子-β1, 白细胞介素-34/集落刺激因子-1受体, 肺纤维化, 上皮-间质转化, CCK 8法, 免疫印迹法, 实时定量聚合酶链反应, A549细胞

Abstract:

Objective To investigate the expression of interleukin（IL)-34/ colony stimulating factor（CSF）-1R in the process of transforming growth factor（TGF）-β1 inducing epithelial-mesenchymal transition (EMT) of human alveolar epithelial cells A549 cells. Methods A549 cells were cultured in vitro. CCK 8 was used to test the influence of the proliferative rate of A549 cells which were stimulated by TGF-β1 at different concentrations and time points. A549 cells were stimulated by 5μg/L TGF-β1 at 0 hour, the 12th hour, the 24th hour, and the 48th hour. Western blotting was adopted to detect changes of the following proteins: α-smooth muscle actin(α-SMA), E-cadherin（E-Cad）, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1). Real-time PCR was adopted to detect changes of the following genes: IL-34 mRNA, CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA. Results TGF-β1 had no significant influence in the proliferation of A549 cells compared with the control group(P>0.05). TGF-β1（5μg/L）stimulated A549 cells at different time point (0 hour, 12, 24, 48 hours), compared with the control group. The epithelial phenotype E-Cad protein was gradually down-regulated(P<0.01), while the mesenchymal phenotype α-SMA protein was gradually up-regulated(P<0.01) and the protein of MMP-2 increased gradually (P<0.01). The protein of MMP-9 increased firstly and then was reduced(P<0.01),the peak was at the 24th hour. The protein of TIMP-1 was firstly transiently increased and then reduced(P<0.01)， the minimum was at the 48th hour. Compared with the control group, the gene of IL-34 mRNA increased gradually (P<0.01), and the genes of CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA increased firstly and then decreased(P<0.01)， which were peaked respectively at the 24th hour, the 24th hour, the 12th hour, respectively. Conclusion In the process of TGF-β1 inducing A549 cells transition，there is accompanied with the expression of IL-34/CSF-1R.

Key words: Transforming growth factor-β1, Interleukin-34/colony stimulating factor-1R, Pulmonary fibrosis, Epithlial-mesenchymal transition, CCK 8, Western blotting, Real-time PCR, A549 cell