代谢综合征基础与临床系列研究——GRGDSP/7E3对动脉粥样硬化内皮细胞MMPs的mRNA表达的影响

目的了解GRGDSP/7E3对基质金属蛋白酶（MMP-2,MMP-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,MMP-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interleukin-1β（IL-1β）,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,而活化状态的血小板能显著诱导这二者的分泌,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）作用后检测发现,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,而静息状态的血小板作用不显著。加入GRGDSP或7E3后,mR-NA的表达降低。结论①活化状态的血小板与IL-1β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（EC）-uPAR和MT1-MMP及上清液中MMP-2mRNA的表达;②GPⅡb/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 GRGDSP/7E3 to Matrix Metalloproteinase of Blood Platelets-endothelial Cells mRNA Express

Objective We evaluated the effects of GRGDSP/7E3 to 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-minute coincubation under cell culture conditions,all platelets were removed by gentle washing.Mter an additional 6 hours of incubation of the endothelial cells,supernatant was aspirated and used for SDS-PAGE zymography and RT- PCR. HUVEC was analyzed by flow cytometry.Results HUVEC was incubated with a-thrombin-activated platelets. SDS- PAGE zymography revealed expression of MMP-2 and MMP-9 was only slightly induced by resting platelets. However, adhesion of activated platelets induced secretion of the 2 MMPs 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 mAbs anti-GPⅡb/Ⅲa （7E3）.HUVEC incubation with α- thrombin-activated platelets induced mRNA expression of MMP-2,MTI-MMP,and uPAR comparable with the control group,but nonstimulated platelets did nothing. Expression of mRNA after administrating GRGDSP or 7E3 decreased greatly. Conclusions These data indicate that platelets can focus matrix-degrading activity to the particular sites of platelet adhe- sion at the vessel wall.GPⅡb/Ⅲa blockade may not only inhibit platelet aggregation at the vulnerable plaque and thereby prevent physical vessel occlusion but also may prevent platelet adhesion mediated inflammatory cascades leading to matrix degradation and plaque rupture.