阻断CD154-CD40信号途径对血小板诱导内皮细胞表达基质金属蛋白酶-1的影响

Effects of blocking CD154-CD40 signal pathway on platelets induced matrix metalloproteinase-1 expression in endothelial cells

目的 :探讨活化血小板及T淋巴细胞经CD15 4分子诱导内皮细胞表达基质金属蛋白酶 - 1(MMP - 1)的功能差异 ;并分析阻断CD15 4 -CD4 0信号途径对血小板诱导内皮细胞表达MMP - 1的影响。方法 :体外分离健康人血小板及淋巴细胞 ,凝血酶 (1U/ml )诱导血小板活化 ,植物血凝素 (10mg/L)和佛波脂 (2 0 μg/L)刺激T淋巴细胞增殖并发生活化。以流式细胞术检测血小板及T淋巴细胞CD15 4表达水平 ,然后使其分别与培养的人脐静脉内皮细胞 (HUVECs)共育。以未经刺激的HUVECs作为阴性对照 ,TNF -α(2 0 0U/ml)刺激的HUVECs作为阳性对照 ,并使用特异性CD15 4及CD4 0阻断单抗 ;逆转录 -聚合酶链式反应测定HUVECs表达MMP - 1mRAN水平 ,底物凝胶电泳酶谱法检测培养基中MMP - 1活性。结果 :以表达同等水平CD15 4的血小板及T淋巴细胞分别作用于HUVECs后 ,均能显著提高MMP - 1mRNA表达 ,同时培养基中MMP - 1活性也明显增强 (P <0 .0 1)。但在诱导血小板或T淋巴细胞活化前加入CD15 4单抗 ,上述效应又都受到明显抑制。同样 ,在活化血小板诱导HUVECs之前 ,如果向培养基中加入CD4 0单抗 ,也能显著降低MMP - 1mRNA表达及其活性 (P <0 .0 1)。结论 :活化血小板及T淋巴细胞表面的CD15 4分子 ,在诱导内皮细胞表达MMP - 1方面具有完全相?

Aim: To explore the functional differences between activated platelets and T lymphocytes in inducing matrix metalloproteinase-1 (MMP-1) expression through CD154 molecule, and investigate the effects of blocking CD154-CD40 signal pathway on platelets induced MMP-1 expression in endothelial cells. Methods: Platelets and lymphocytes were isolated from healthy volunteers, and thrombin (1 U/ml) was used in vitro to activate platelets. At the same time, phytha- emagglutinin (10 mg/L) and phorbol myristate acetate (20 μg/L) were jointly added into suspension to acquire proliferative and activated T lymphocytes. After determined the levels of CD154 expression by flow cytometry technique, activated platelets and T lymphocytes were co-incubated with cultured human umbilical vein endothelial cells (HUVECs), respectively. With non-stimulated HUVECs as negative control and TNF-α(200 U/ml) stimulated HUVECs as positive control, MMP-1 mRNA expression in HUVECs was assayed by reverse transcription-polymerase chain reaction(RT-PCR) and MMP-1 activity in supernatant was analyzed by using substrate gel electrophoresis zymography. Results: Activated platelets and T lymphocytes, which express same intensity of CD154, equally raised MMP-1 mRNA expression in HUVECs following increased activities of MMP-1 in supernatant (P<0.01). But these effects could be attenuated by CD154 monoclonal antibody added into media before platelets or T lymphocytes being induced. Moreover, the stimulation effects of activated platelets on MMP-1 expression in HUVECs were also markedly inhibited by CD40 monoclonal antibody added into media (P<0.01). Conclusion: CD154 expressed on activated platelets and T lymphocytes performs the same biological function in up-regulating MMP-1 expression in endothelial cells, and blocking CD154-CD40 signal pathway may effectively inhibit the MMP-1 expression induced by platelets.