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目的探讨组织型金属蛋白酶抑制剂4(tissue inh ib itors of m atrix m etalloprote inases-4,TIMP-4)对基质金属蛋白酶(MMP)活性的作用,观察其对血管组织胶原含量的影响。方法实验动物分3组,单纯损伤组,损伤后转染AdGFP组和损伤后转染AdGFP-4组。以复制缺陷型腺病毒为载体,将TIMP-4转移到培养的大鼠血管平滑肌细胞中,用凝胶电泳酶谱分析和反向酶谱分析法检测TIMP-4对MMP活性的影响;制备大鼠颈总动脉球囊损伤模型并转染TIMP-4基因,检测TIMP-4对血管组织MMP活性及对胶原含量的影响。结果转染AdTIMP-4可使培养的血管平滑肌细胞MMP-2的活性降低至对照组(未转基因组和转染AdGFP组)的26%。动物实验中血管损伤后转染AdTIMP-4组与单纯损伤组比较,其活化形式的MMP-2几乎完全被抑制;损伤后转染AdTIMP-4组与转染AdGFP组比较,血管新生内膜的面积和细胞均减少,细胞内膜天狼星红含量的吸光度(55 030±5899)明显低于转染AdGFP组(91 530±7729,P<0.05),并不促进血管组织胶原沉积(每个细胞的胶原含量135±11 vs 118±13,P>0.05)。结论TIMP-4通过改变MMP/TIMP的平衡调节血管组织胶原代谢,在血管损伤后修复过程中发挥重要作用。
Objective To investigate the effect of tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) on the activity of matrix metalloproteinase (MMP) and to observe its effect on collagen content in vascular tissue. Methods The experimental animals were divided into 3 groups: the simple injury group, the AdGFP group and the AdGFP-4 group. TIMP-4 was transfected into cultured rat vascular smooth muscle cells using replication-deficient adenovirus as a vehicle, and the effect of TIMP-4 on MMP activity was examined by gel electrophoresis and reverse-phase zymography; TIMP-4 gene was transfected into rat common carotid artery balloon injury model and the effect of TIMP-4 on MMP activity and collagen content in vascular tissue was detected. Results AdTIMP-4 transfection reduced the activity of MMP-2 in cultured vascular smooth muscle cells to 26% of the control group (untransfected group and transfected AdGFP group). Compared with the simple injury group, the activated form of MMP-2 in AdTIMP-4 group was almost completely inhibited after transfection in AdTIMP-4 group. Compared with AdGFMP-4 group, the expression of intima (55 030 ± 5899) was significantly lower than that of transfected AdGFP group (91 530 ± 7729, P <0.05), and did not promote the deposition of vascular collagen (per cell Collagen content 135 ± 11 vs 118 ± 13, P> 0.05). Conclusion TIMP-4 regulates the collagen metabolism of vascular tissue by changing the balance of MMP / TIMP and plays an important role in the repair process after vascular injury.