TIMP-2、MMP-2和MT1-MMP在活化肝星状细胞中的表达及对细胞外基质合成分泌的影响

目的 研究金属蛋白酶组织抑制剂-2(TIMP-2)、基质金属蛋白酶-2(MMP-2)和膜型基质金属蛋白酶-1(MT1-MMP)在活化肝星状细胞(HSCs)中的表达,观察其对细胞外基质(ECM)合成分泌的影响.方法 原代分离培养大鼠HSCs活化后,分别给予40～160 pmol化学合成经修饰抗TIMP-2 siRNA进行干预,检测培养细胞上清液透明质酸(HA)、Ⅲ型前胶原(PCⅢ)和羟脯氨酸(Hyp)的含量,采用荧光实时定量PCR法检测TIMP-2、MMP-2、MT1-MMP、MMP-13、COL Ⅰ和COL Ⅲ mRNA的表达,western印迹检测TIMP-2、MT1-MMP和MMP-13蛋白表达及明胶酶谱法检测MMP-2蛋白表达.结果 应用化学合成经修饰抗TIMP-2 siRNA后,TIMP-2、MMP-2、MT1-MMP、COL Ⅰ和COL Ⅲ的表达明显降低,而MMP-13的表达则明显增加,培养细胞上清液中HA、PCⅢ和Hyp的含量也明显减少.结论 TIMP-2通过MT1-MMP介导MMP-2的活化,抑制TIMP-2的表达,MT1-MMP和MMP-2的表达随之降低,而HSCs合成分泌ECM也相应减少.

Expressions of TIMP-2, MMP-2 and MT1-MMP in activated hepatic stellate cells and their effects on the synthesis and secretion of extracellular matrix

Objective To investigate the relationship among tissue inhibitors of metalloproteinase-2 (TIMP-2), matrix metalloproteinase-2(MMP-2) and membrane type 1 matrix metalloproteinase (MT1-MMP) and study their effects on the synthesis and secretion of extracellular matrix (ECM). Methods HSCs were isolated from Sprague-Dawley rats by collagenase and pronase E perfusion of liver from through portal vein and by 12% Nycodenz gradient centrifugation. After activation of HSCs, HSCs were treated with the quantity ranging from 40 pmol to 160 pmol of chemically modified small interfering RNA (siRNA) targeting TIMP-2. The levels of hyaluronic acid (HA), procolagen type III (PCIII) and hydroxyproline (Hyp) in supernatant were measured. Expressions of TIMP-2 mRNA, MMP-2 mRNA, MT1-MMP mRNA, MMP-13 mRNA, COL I mRNA and COL III mRNA were detected by quantitative real-time PCR. Protein levels of TIMP-2, MT1-MMP and MMP-13 were evaluated by western blotting, and the content of MMP-2 was examined by gelatin zymography. Results The levels of HA, PCIII and Hyp in three groups treated with siRMA was decreased markedly compared with that in normal group. The expression of TIMP-2, MMP-2, MT1-MMP , COL I and COL III in three treated groups were significantly lower than that in normal group, but the expression of MMP-13 was significantly higher than that in normal group. Conclusion TIMP-2 mediated by MT1-MMP regulated MMP-2 activation. The siRNA targeting TIMP-2 reduces the expression of TIMP-2, which associated with down-regulation of MT1-MMP and MMP-2, could suppress the synthesis and secretion of ECM in activated HSCs.