基础医学与临床 ›› 2014, Vol. 34 ›› Issue (12): 1650-1654.

• 研究论文 • 上一篇    下一篇

重组的脂联素调节肝细胞系L02基质金属蛋白酶-9表达及活性

陶冬英1,王云2,倪晶晶1,杨菊林1,范任华3   

  1. 1. 宁波卫生职业技术学院
    2. 宁波二院外科
    3. 长沙市中心医院检验科
  • 收稿日期:2014-02-27 修回日期:2014-06-24 出版日期:2014-12-05 发布日期:2014-11-25
  • 通讯作者: 陶冬英 E-mail:764897579@qq.com
  • 基金资助:
    浙江省教育厅课题项目;国家青年科学基金项目

Recombinant adiponectin regulates hepatocyte L02 matrix metalloproteinase-9 expression and activity

  • Received:2014-02-27 Revised:2014-06-24 Online:2014-12-05 Published:2014-11-25

摘要: 目的 观察重组脂联素对肝细胞系L02基质金属蛋白酶-9表达及活性的影响,探讨脂联素在肝纤维化中的作用。方法 细胞分未转染对照组、转染空载体组、整合了adi-pEGFP-C1的L02细胞三组。体外扩增脂联素adiponectin mRNA全长,通过双酶切、插入及连接等基因克隆方法构建真核表达载体adi-pEGFP-C1。然后通过基因转染方法,将adi-pEGFP-C1转染到肝细胞系L02细胞中。通过G418压力筛选出adi-pEGFP-C1/L02稳定细胞系,用基因、蛋白以及蛋白酶谱等技术检测基质金属蛋白酶MMP-9的基因水平、蛋白水平以及酶的活性水平。结果 adi-pEGFP-C1构建成功,并成功筛选到L02稳定细胞系adi-pEGFP-C1/L02,与空白对照组相比,MMP-9 mRNA表达水平上调(P<0.01),MMP-9蛋白水平和活性均上调(P<0.05)。结论 脂联素通过调节MMP-9的表达及活性可能影响肝纤维化进程。

关键词: 关键词: 脂联素, 肝纤维化, 肝细胞L02, 基质金属蛋白酶-9

Abstract: Objective To observe the effect of the expression and activity on the hepatocyte L02 matrix metalloproteinase-9 by the recombinant adiponectin, and investigate the effect of adiponectin in liver fibrogenesis liver. Methods The cells were divided into control groups, pEGFP-C1/L02 groups and adi-pEGFP-C1/L02 groups.The full-length of human adiponectin mRNA was amplified by reversal transcriptional polymerase chain reaction (RT-PCR) in vitro. Eukaryotic expression vector of adiponectin (adi-pEGFP-C1) was constructed by double enzyme, insertion, connection and gene cloning method and so on. The adi-pEGFP-C1 vector was transfected into the liver cell line L02 cells by the gene transfection method with lipofatmine2000. The stable cell lines of adi-pEGFP-C1/L02 were screened by G418. The protein, mRNA and activity of matrix metalloproteinases-9 (MMP-9) were detected by Western-blotting, RT-PCR and gel zymography .Results The recombinant vectors adi-pEGFP-C1 was constructed and the stable cell lines of adi-pEGFP-C1/L02 were screened successfully. The mRNA and protein expression levels of MMP-9 were increased by RT-PCR (P <0.01) and Western-blotting(P<0.05)compared with the control groups; The activity of MMP-9 was increased by gel zymography (P <0.05) compared with the control groups. Conclusions Activation of adiponectin may probably affect the process of liver fibrosis by up-regulated the expression and activity of MMP-9.

Key words: Key words: Adiponectin, hepatic fibrosis, L02 hepatocytes, matrix metalloproteinases-9

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