血小板膜糖蛋白对动脉粥样硬化内皮细胞基质金属蛋白酶的影响

目的：了解血小板膜糖蛋白（GP）Ⅱb／Ⅲa对基质金属蛋白酶（MMP-2，9）mRNA表达的影响。方法：体外培养人脐静脉内皮细胞，分别用酶谱法和逆转录聚合酶链式反应（RT-PCR）检测MMP-2，-9活性和mRNA表达。待细胞融合成单层后，分别加入无血清培养基，静息期血小板，活化后血小板及白细胞介素-1β（IL-β），共同培养60min后，洗去血小板，内皮细胞继续培养6h。上清液及细胞分别用于酶谱法检测MMP-2和MMP-9活性，以及RT-PCR分析mRNA表达。结果：内皮细胞与活化血小板共同培养后，酶谱法显示：与对照组相比，未活化的血小板轻微诱导MMP-2和MMP-9分泌（P〈0．05），而活化状态的血小板能显著诱导这二者的分泌（P〈0．01），与IL-1β的作用相当；而且还可以见到62kDaMMP-2活化形式。加入阻断剂GRGDSP以及GPⅡb／Ⅲa单克隆抗体（7E3）后检测发现，MMP-2和MMP-9分泌减少。与活化血小板共同培养后的内皮细胞表面表达uPAR和MT1-MMP以及上清液中MMP-2的mRNA与对照组相比明显增高（P〈0．01），而静息状态的血小板作用不显著。加入GRGDSP或7E3后，mRNA的表达降低。结论：（1）活化状态的血小板与IL-8的作用相当，能显著诱导MMP-2和MMP-9分泌；能明显增高内皮细胞uPAR和MT1-MMP及上清液中MMP-2mRNA的表达；（2）GPⅡb／Ⅲa阻断剂可能抑制血小板在不稳定斑块部位聚集、粘附等一系列炎症反应。

Influence of GP Ⅱb/Ⅲa on matrix metalloproteinase of blood platelets-endothelial cells Mrna express

Objective： To study the effects of GPⅡb/Ⅲa on matrix metalloproteinase of blood platelets-endothelial cells mRNA express. Methods： The HUVECs was cultured in vitro. When the cells were sub-confluent, add serum free medium, resting platelets,α-thrombin-stimulated platelets and interleukin-1β （IL-1β） into the 24 well plate respectively. After 60 min co-incubation under cell culture conditions, all platelets were removed by gentle washing. After an additional 6 hours of incubation of the endothelial cells, supernatant was aspirated and detected the activity of MMP-2 and MMP-9 by SDS-PAGE zymography and detected the mRNA by RT-PCR. HUVEC was analyzed by flow cytometry. Results： HUVEC was incubated with α-thrombin-activated platelets. SDS-PAGE zymography revealed expression of MMP-2 and MMP-9 was only slightly induced by resting platelets （P〈0.05）. However, activated platelets significantly induced secretion of the 2 MMPs （P〈0.01） to a comparable extent as achieved by IL-1β. In addition, a second, lower band at 66kDa revealed MMP-2 activation on adhesion of activated platelets. Endothelial expression of MMP-2 and MMP-9 was significantly restrained by GRGDSP and monoclonal antibody of anti-GPⅡb/Ⅲa （7E3） . HUVEC incubation with activated platelets induced mRNA significantly expression of MMP-2, MT1 -MMP and uPAR （P〈0.01） comparable with the control group, but nonstimulated platelets did nothing. Expression of mRNA greatly decreased after administrating GRGDSP or 7E3. Conclusion： Activated platelets significantly induced secretion of the 2 MMPs to a comparable extent as achieved by IL-1β, and can increase mRNA expression of MMP-2, MT1- MMP, and uPAR. GPⅡb/Ⅲa blockade may not only inhibit platelet aggregation at the vulnerable plaque, but also may prevent platelet adhesion mediated inflammatory cascades.