过表达Raf激酶抑制蛋白(RKIP)抑制人肝星状细胞增殖

Over-expression of Raf kinase inhibitor protein(RKIP) inhibits the proliferation of LX-2 human hepatic stellate cells

目的研究Raf激酶抑制蛋白(RKIP)对LX-2人肝星状细胞增殖的影响。方法用重组质粒pc DNA3.1-RKIP转染LX-2细胞,G418筛选并培养稳定转染的细胞。MTT法和集落形成试验检测过表达RKIP对LX-2细胞增殖、集落生成的影响;Western blot法检测RKIP、α平滑肌肌动蛋白(α-SMA)、1型胶原蛋白(Col1)、基质金属蛋白酶1(MMP-1)、MMP-2及胞外信号调节激酶/丝裂原激活蛋白激酶(ERK/MAPK)信号通路相关蛋白的表达水平。结果与对照细胞相比,过表达RKIP明显抑制LX-2细胞增殖和集落形成,下调Col1、α-SMA、MMP-1和MMP-2蛋白表达,降低ERK/MAPK磷酸化水平。结论过表达RKIP抑制LX-2细胞的增殖,其机制与抑制ERK/MAPK信号通路有关。

Objective To investigate the effect of Raf kinase inhibitor protein（RKIP） over-expression on the proliferation of LX-2 human hepatic stellate cells.Methods Recombinant plasmid pc DNA3.1-RKIP was transfected into LX-2 cells.G418 was used to screen and culture stably infected cells.MTT assay and colony formation assay were used to examine the effect of RKIP over-expression on cell proliferation and colony formation,respectively.Western blotting was performed to assess the expressions of RKIP,α-smooth muscle actin（α-SMA）,type 1 collagen（Col1） and matrix metalloproteinase 1（MMP-1）and MMP-2 as well as extracellular signal-regulated kinases/mitogen-activated protein kinase（ERK/MAPK） signaling pathway-related proteins.Results Compared with the control cells,RKIP over-expression significantly inhibited LX-2 cell proliferation and colony formation,and reduced the protein expressions of Col1,α-SMA,MMP-1 and MMP-2.Moreover,RKIP over-expression remarkably inhibited the phosphorylation of ERK/MAPK.Conclusion Over-expressed RKIP inhibits LX-2 cell proliferation and the mechanism is related to the inhibition of ERK/MAPK signaling pathway.