脑源性神经营养因子对细胞外蛋白水解酶激活作用的研究

Effects of brain-derived neurotrophic factor on extracellular proteolytic enzymes

目的:研究脑源性神经营养因子(BDNF)对细胞外蛋白水解酶表达和激活作用的影响。方法:体外分离并培养人脐静脉内皮细胞(HUVEC),RT-PCR法检测HUVEC基质金属蛋白酶MMP-2、MMP-9和基质金属蛋白酶组织抑制剂TIMP-1、TIMP-2mRNA的表达,明胶酶谱检测MMP-2和MMP-9蛋白酶活性,纤维蛋白酶谱检测尿激酶型纤溶酶原激活剂(uPA)蛋白酶活性,Western blotting检测uPA、纤溶酶原激活剂抑制剂(PAI)、TIMP-1及TIMP-2表达。结果:在对HUVEC增殖无明显促进作用的浓度范围内,BDNF可促进无血清培养的HUVEC MMP-2和MMP-9mRNA表达,并可促进MMP-2和MMP-9酶原的激活产生活性明胶酶,BDNF对TIMP-1和TIMP-2的表达无明显影响。BDNF以浓度和时间依赖性方式上调HUVEC uPA和PAI-1的表达,并可促进uPA的活性。结论:BDNF可激活MMPs和uPA/PAI相关的蛋白级联。

AIM： To investigate the effects of brain -derived neurotrophic factor （BDNF） on extracellular proteolytic enzymes including matrix-metaUoproteinases （MMPs） and serine proteases, in particular, the urokinase - type phisminogen activator （uPA） - plasmin system in a human umbilical vein endothelial cell （HUVEC） model. METHODS： The HUVEC was activated with different doses of BDNF （25 -200μg/L） for different time period （6 -48 h）. Reverse transcriptase - polymerase chain reaction （ RT - PCR） was used to assay MMP - 2, MMP - 9, TIMP - 1, and TIMP - 2 mRNA in HUVEC. The cultured conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography, respectively, uPA, PAl - I, TIMP - 1, and TIMP - 2 were quantified by Western blotting analysis. RESULTS： The stimulation of serum - starved HUVECs with BDNF caused marked increase in MMP - 2 and MMP - 9 mRNA expression and induced the pro -MMP -2 and pro -MMP- 9 activation without significant differences in proliferation. However, BDNF had no effect on ＂TIMP - 1 and ＂TIMP - 2 production. BDNF increased uPA and PAl - 1 production in a dose dependent manner up to 100μg/L, while effects of 200μg/L were approximately equal to those of 100μg/L. BDNF stimulated uPA and PAI - 1 production beyond that in control cultures from 12 h until 48 h after BDNF addition. Protease activity for uPA was also increased by BDNF in a dose dependent manner. CONCLUSION： BDNF activates MMP and uPA/PAI - 1 proteolytic network in HUVEC.