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.doc

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 　　作者：高阳△,陈思娇*,熊盈△,胡怡,魏敏　　作者单位：中国医科大学附属第一医院老年医学研究室，辽宁 沈阳 110001;中国人民解放军第202医院;中国医科大学医学分子生物学研究所。 △中国医科大学硕士研究生 　　【摘要】目的了解血小板膜糖蛋白(GP)Ⅱ b/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60 min后，洗去血小板，内皮细胞继续培养6 h。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(P0. 05)，而活化状态的血小板能显著诱导这二者的分泌(P0. 01)，与IL-1Β的作用相当;而且还可以见到62 kDaMMP-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(P0. 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-Β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-2 mRNA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。 　　【关键词】 血小板膜糖蛋白,动脉粥样硬化,基质金属蛋白酶 　　Abstract：Objective:To study the effects of GP Ⅱb/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-lΒ (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 SDSzymography and detected the mRNA by RT-PCR.HUVEC was analyzed by flow cytometry.Results:HUVEC was incubated with Α-thrombin|activated platelets. SDSzymography revealed expression of MMP-2 and MMP-9 was only slightly induced by resting platelets(P0. 05).However, activated platelets significantly induced secretion of the 2 MMPs(P0. 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