中国医科大学学报

中国医科大学学报

中国医科大学学报 ›› 2012, Vol. 41 ›› Issue (6): 481–484.

• 基础医学 •    下一篇

EMAP-Ⅱ对紧密连接相关蛋白occludin和ZO-1相互作用的调节和机制

王佐周1, 刘丽波2,薛一雪2,刘云会3   

  1. (中国医科大学1. 基础医学院病理学教研室,沈阳 110001;2. 基础医学院神经生物学教研室,沈阳 110001;3. 附属盛京医院神经外科,沈阳 110004)
  • 收稿日期:2012-09-25 修回日期:2012-09-25 出版日期:2012-06-20 发布日期:2012-09-27
  • 基金资助:
    国家自然科学基金资助项目(30973079,81072056,30872656,30670723);辽宁省教育厅科学基金(L2010569);沈阳市科学基金(F10-205-1-22);教育部专项科研基金(20092104110015,20102104120023,20102104110009)

Regulation and Mechanism of EMAP-Ⅱ on the Interaction of Tight Junction Associated Protein Occludin and ZO-1

WANG Zuo-zhou1, LIU Li-bo2, XUE Yi-xue2, LIU Yun-hui3   

  1. (1. Department of Pathology, College of Basic Medical Science, China Medical University, Shenyang 110001, China; 2. Department of Neurobiology, College of Basic Medical Science, China Medical University, Shenyang 110001, China; 3. Department of Neurosurgery, Shengjing Hospital, China Medical University, Shenyang 110004, China)
  • Received:2012-09-25 Revised:2012-09-25 Online:2012-06-20 Published:2012-09-27

摘要: 目的 研究内皮—单核细胞激活多肽Ⅱ(EMAP-Ⅱ)对血肿瘤屏障(BTB)紧密连接相关蛋白occludin和ZO-1分布和相互作用的影响,以及蛋白激酶C(PKC)活性的变化,探讨PKC在EMAP-Ⅱ开放紧密连接中的作用。方法 提取和培养大鼠脑微血管内皮细胞,建立体外BTB模型;实验分为7组,即模型组、EMAP-Ⅱ 0.5 h组、EMAP-Ⅱ 1 h组、EMAP-Ⅱ 2 h组、EMAP-Ⅱ 3 h组、EMAP-Ⅱ 6 h组和EMAP-Ⅱ 12 h组。应用Milicell-ERS系统检测体外BTB模型跨内皮细胞阻抗值的变化;双重免疫荧光检测内皮细胞中紧密连接相关蛋白occludin和ZO-1 的分布和定位;免疫共沉淀检测内皮细胞中occludin和ZO-1的结合水平;PKC活性检测试剂盒检测体外BTB模型内皮细胞中总PKC的活性。结果 与模型组相比,EMAP-Ⅱ显著降低了体外BTB模型的跨内皮细胞阻抗值,增加了BTB通透性;在模型组,紧密连接相关蛋白occludin和ZO-1 在内皮细胞边缘呈连续分布,并且二者存在共定位,EMAP-Ⅱ能够使occludin和ZO-1 在内皮细胞边缘的分布减少,二者的共定位减弱;免疫共沉淀结果也显示EMAP-Ⅱ能够减弱occludin和ZO-1的结合;上述作用在EMAP-Ⅱ作用 1 h时效果最显著(P<0.01);同时发现EMAP-Ⅱ作用后,BTB模型内皮细胞中PKC的活性显著升高,在EMAP-Ⅱ作用 1 h时达峰值(P<0.01)。结论 EMAP-Ⅱ能够调节紧密连接相关蛋白occludin和ZO-1在内皮细胞的相互作用,开放紧密连接;PKC可能是EMAP-Ⅱ影响occludin和ZO-1相互作用、调节紧密连接的机制之一。

关键词: 内皮—单核细胞激活多肽Ⅱ, occludin, ZO-1, 蛋白激酶C, 血肿瘤屏障

Abstract: Objective To investigate the effect of endothelial-monocyte activating polypeptide Ⅱ (EMAP-Ⅱ) on the distribution and interaction of the tight junction associated proteins occludin and ZO-1 as well as the change of the activity of protein kinase C (PKC), and further analyze the action of PKC in the process of EMAP-Ⅱ opening the tight junction. Methods The blood-tumor barrier (BTB) models were established by extracting and culturing the rat brain microvascular endothelial cells (BMEC). Subjects were divided into 7 groups: model group, EMAP-Ⅱ 0.5 h group; EMAP-Ⅱ 1 h group, EMAP-Ⅱ 2 h group, EMAP-Ⅱ 3 h group, EMAP-Ⅱ 6 h group and EMAP-Ⅱ 12 h group. The Milicell-ERS system was used to measure the transendothelial electrical resistance (TEER). The distribution and location of occludin and ZO-1 were detected by double-immunofluorescence assay. Immunoprecipitation assay was used to test the interaction of occludin and ZO-1 in BMEC. The activity of total PKC was detected by PKC assay kit. Results In contrast to model group, EMAP-Ⅱ groups showed that EMAP-Ⅱ obviously decreased the TEER of in BTB model in vitro and increased its permeability. In model group, the tight junction associated proteins occludin and ZO-1 was localized to the cell boundaries with continuous distribution and both of them presented co-location. After EMAP-Ⅱ administering, occludin and ZO-1 were discontinuously distributed in the cellular boundaries and the co-location of them was attenuated. The result of immunoprecipitation showed that EMAP-Ⅱ could attenuate the combination of occludin and ZO-1. The above actions were most obvious when EMAP-Ⅱhad been administered for 1h(P<0.01). Meanwhile, the activity of total PKC was increased significantly after EMAP-Ⅱ administering and the peak turned up at EMAP-Ⅱ action 1 h (P<0.01). Conclusion EMAP-Ⅱ could regulate the interaction of tight junction associated proteins occludin and ZO-1 in BMEC and open the tight junction. PKC might be one of the mechanisms of EMAP-Ⅱ regulating the interaction of occludin and ZO-1 and opening the tight junction.

Key words: EMAP-Ⅱ, occludin, ZO-1, protein kinase C, blood-tumor barrier

中图分类号: 

  • 中图分类号 R 341.6 文献标志码 A 文章编号 0258-4646(2012)06-
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