文章摘要
张丹丹,夏文龙,刘 强,张大伟,徐晋妉,吴恒芳,陈相健,杨 笛.OX40/F0X40L逆向信号对小鼠主动脉内皮细胞MMP-9表达及机制的研究[J].南京医科大学学报,2012,(1):20~25
OX40/F0X40L逆向信号对小鼠主动脉内皮细胞MMP-9表达及机制的研究
The effects and mechanism of OX40/OX40L reverse signaling on the expression of MMP-9 in mouse aortic endothelial cells
投稿时间:2011-07-27  
DOI:10.7655
中文关键词: OX40配体  钙离子  基质金属蛋白酶-9  内皮细胞
英文关键词: OX40 ligand  [Ca2+]i  matrix metalloproteinase-9  endothelial cells
基金项目:国家自然科学基金项目(30772781,30770894)
作者单位
张丹丹 南京医科大学第一附属医院心内科,江苏 南京 210029 
夏文龙 南京医科大学第一附属医院心内科,江苏 南京 210029 
刘 强 南京医科大学第一附属医院心内科,江苏 南京 210029 
张大伟 南京医科大学第一附属医院心内科,江苏 南京 210029 
徐晋妉 南京医科大学第一附属医院心肺疾病研究所,江苏 南京 210029 
吴恒芳 南京医科大学第一附属医院心肺疾病研究所,江苏 南京 210029 
陈相健 南京医科大学第一附属医院心肺疾病研究所,江苏 南京 210029 
杨 笛 南京医科大学第一附属医院心内科,江苏 南京 210029 
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中文摘要:
      目的:探讨OX40配体(OX40 ligand,OX40L)逆向通路激活对培养的小鼠主动脉内皮细胞基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达及活性的作用?方法:分离与培养8~10周龄C57BL/6J小鼠主动脉内皮细胞,传代至第2代,用可溶性OX40(sOX40)?OX40L单克隆抗体(anti-OX40L)和钙离子螯合剂bapta-AM刺激内皮细胞,RT-PCR和Western blot方法检测内皮细胞MMP-9的mRNA和蛋白表达水平,明胶酶谱法检测内皮细胞培养上清MMP-9活性,应用钙离子成像方法观察内皮细胞胞浆游离钙离子浓度([Ca2+]i)的改变?结果:与对照组相比,可溶性OX40(0.1 mg/L)刺激后内皮细胞MMP-9的mRNA及蛋白表达水平均显著增高,内皮细胞培养上清MMP-9活性明显增高,内皮细胞胞浆[Ca2+]i也明显增高?分别应用可溶性OX40L单克隆抗体(0.1 mg/L)和Bapta-AM(20 μmol/L)预处理培养内皮细胞后,由可溶性OX40引起的内皮细胞基因和蛋白表达水平明显降低,内皮细胞培养上清MMP-9活性明显下降?结论:内皮细胞OX40L逆向信号的激活可以通过调节胞内的游离钙离子水平参与内皮细胞中MMP-9表达的调控,这可能是OX40/OX40L逆向信号通路参与动脉粥样硬化的发生?发展的机制之一?
英文摘要:
      Objective: To investigate the effects of OX40 ligand(OX40L) reverse signaling on the expression and activity of matrix metalloproteinase-9 (MMP-9) in the cultured mouse aortic endothelial cells (MAECs). Methods: Aortic endothelial cells were isolated from C57BL/6J mice aged 8 to 10 weeks and cultured to the second passage for experiments. The cultured MAECs were divided into 4 groups:control group,sOX40 group,sOX40+anti-OX40L group and sOX40+Bapta-AM group,and stimulated with DMEM,soluble OX40,anti-OX40L monoclonal antibody and calcium chelator Bapta-AM,respectively. The mRNA and protein expressions of MMP-9 were determined by RT-PCR and Western blot. The activity of MMP-9 was assayed by zymography. The intracellular calcium ([Ca2+]i) in MAECs was detected by calcium imaging using fura-2. Results: A significant increase in intracellular calcium level of MAECs was observed after stimulation with soluble OX40 (0.1 mg/L) for 48 hours compared to the control,and MMP-9 mRNA and protein levels were also markedly elevated. The activity changes of MMP-9 in cell culture supernatant was consistent with that of intracellular level. Notably,these elevations can be blocked by soluble anti-OX40L monoclonal antibody(0.1 mg/L) or Bapta-AM(20 μmod/L). Conclusion:The stimulation of OX40L reverse signaling can activate the expression of MMP-9 on both mRNA and protein levels through the elevation of intracellular calcium of MAECs,suggesting that the OX40/OX40L signaling pathway may be one of the pathways contributing to the MMP-9 elevation in progression of atherosclerosis.
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