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中华普通外科学文献(电子版) ›› 2014, Vol. 08 ›› Issue (05) : 353 -358. doi: 10.3877/cma.j.issn.1674-0793.2014.05.005

所属专题: 文献

论著

结缔组织生长因子通过ERK1/2和PI3K信号通路促进大鼠肝星状细胞迁移和基质金属蛋白酶2表达
甄茂川1, 刘平果1, 赵一麟1, 吴绍峰1, 俞可克1, 尹震宇1,()   
  1. 1. 361004 厦门大学附属中山医院肝胆外科
  • 收稿日期:2014-05-06 出版日期:2014-10-01
  • 通信作者: 尹震宇
  • 基金资助:
    国家自然基金资助项目(30872484); 福建省医学创新基金资助项目(2009-CXB-56)

CTGF promoting migration and MMP-2 expression via ERK1/2 and PI3K pathway in rat hepatic stellate cells

Maochuan Zhen1, Pingguo Liu1, Yilin Zhao1, Shaofeng Wu1, Keke Yu1, Zhenyu Yin1,()   

  1. 1. Department of Hepatobiliary Surgery, Xiamen University Zhongshan Hospital, 361004 Xiamen, China
  • Received:2014-05-06 Published:2014-10-01
  • Corresponding author: Zhenyu Yin
  • About author:
    Corresponding author: Yin Zhenyu, Email:
引用本文:

甄茂川, 刘平果, 赵一麟, 吴绍峰, 俞可克, 尹震宇. 结缔组织生长因子通过ERK1/2和PI3K信号通路促进大鼠肝星状细胞迁移和基质金属蛋白酶2表达[J/OL]. 中华普通外科学文献(电子版), 2014, 08(05): 353-358.

Maochuan Zhen, Pingguo Liu, Yilin Zhao, Shaofeng Wu, Keke Yu, Zhenyu Yin. CTGF promoting migration and MMP-2 expression via ERK1/2 and PI3K pathway in rat hepatic stellate cells[J/OL]. Chinese Archives of General Surgery(Electronic Edition), 2014, 08(05): 353-358.

目的

研究结缔组织生长因子(CTGF)对大鼠肝星状细胞(HSC)迁移及基质金属蛋白酶2(MMP-2)表达及活化的影响。

方法

分别应用RT-PCR和Western blotting法检测MMP-2表达,明胶酶谱法检测MMP-2的活性;运用Western blotting法检测ERK1/2和Akt的磷酸化水平。运用Transwell小室检测肝星状细胞迁移能力。

结果

CTGF呈剂量依赖性促进大鼠HSC中MMP-2mRNA和蛋白表达;同时CTGF能够促进HSC迁移,MMP-2特异性抑制剂能够抑制CTGF的促迁移作用。ERK1/2和PI3K抑制剂能够显著抑制CTGF诱导的MMP-2表达以及CTGF促进HSC的迁移作用。

结论

MMP-2表达和活性的增强在CTGF促进HSC迁移过程中发挥着重要作用,ERK1/2和PI3K信号通路在CTGF促进MMP-2表达发挥关键作用。

Objective

To identify the role of connective tissue growth factor (CTGF) in hepatic stellate cell (HSC) migration and its effects on gelatinase matrix metalloproteinase-2 (MMP-2) expression and activation.

Methods

The expression of MMP-2 was assessed by RT-PCR and Western blotting analyses. MMP-2 activity was evaluated by zymography. ERK1/2 and Akt phosphorylation were determined by Western blotting analysis. HSC migration was performed using Transwell cell culture chambers.

Results

The expression of MMP-2 mRNA and protein in HSCs were substantially increased by CTGF treatment in a dose-dependent manner. In addition, CTGF could promote HSC migration, and the addition of MMP-2-specific inhibitor impaired this phenomenon. To study the intracellular signaling pathways involved in CTGF-induced MMP-2 expression, we evaluated the effects of different kinase inhibitors. The inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3-kinase (PI3K) abrogated CTGF-elicited MMP-2 upregulation and significantly reduced CTGF-induced invasiveness of HSC.

Conclusion

MMP-2 plays an important role in the invasive effects of CTGF on HSC. ERK1/2 and PI3K are the main signals involved in CTFG-mediated MMP-2 expression.

图1 CTGF促进HSC迁移(苏木素染色10×10)。P<0.05 vs对照组;P<0.05 vs CTGF(100μg/L)处理组。CTGF对HSCs侵袭的促进作用呈剂量依赖性,加用特异性的MMP-2抑制剂I(OA-Hy)后,CTGF诱导的HSC侵袭作用几乎完全被抑制
图2 CTGF呈剂量依赖性促进HSCs MMP-2的活化P<0.05 vs对照组。A.培养液明胶酶谱结果显示,未经刺激的HSCs培养液呈现72kD的明胶降解带,代表pro-MMP-2;加入CTGF刺激24h后,pro-MMP-2表达增加;并且,可见一强度稍弱的66kD条带,即活化的MMP-2。B.浓度分别为1,10,25,50μmol/L的CTGF诱导的MMP-2活化的呈剂量依赖性
图3 CTGF促进HSCs pro-MMP-2的表达。P<0.05 vs对照组;HSCs加入不同浓度的CTGF作用24h后,结果同明胶酶谱结果相一致,MMP-2mRNA和蛋白表达上调
图4 CTGF(50ng/mL)对HSCs ERK1/2和Akt磷酸化的影响,CTGF(50μg/L)刺激5min后ERK1/2磷酸化显著增强(图4A),相同浓度的CTGF刺激5min后Akt磷酸化亦显著增强(图4B)
图5 Wortmannin和PD98059抑制CTGF诱导的MMP-2表达。P<0.05 vs对照组;P<0.05 vs CTGF(100μg/L)处理组。Wortmannin (50nmol/L)能够显著抑制CTGF诱导的MMP-2表达;PD98059(10μmol/L)亦能够显著抑制CTGF诱导的MMP-2表达
图6 Wortmannin和PD98059抑制CTGF促HSCs迁移作用。P<0.05 vs对照组;P<0.05 vs CTGF(100μg/L)处理组。运用PD98059(10μmol/L)预处理30min后,CTGF诱导的细胞迁移被显著抑制;运用Wortmannin(25nmol/L)预处理后,亦能显著抑制CTGF诱导的细胞迁移
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