【摘要】 膜联蛋白Ⅱ (annexinⅡ )是纤溶酶原和组织型纤溶原激活物的共同受体。本研究旨在探讨annexinⅡ引起急性白血病及其它肿瘤患者原发性纤溶亢进的分子病理机制 ,阐明annexinⅡ促纤溶活性 ,为探讨annexinⅡ在凝血障碍中的作用提供理想的载体模型。用逆转录聚合酶链反应 (RT PCR)扩增annexinⅡ基因片段 ,经纯化及连接 ,用脂质体转染入HL 60细胞中表达 ;用多光子激发的共聚焦显微镜观察其表达产物的胞内分布及结构特性 ;用流式细胞术及Western印迹对转染的细胞表达进行定量及定性分析 ,并揭示其对纤溶酶活性的影响。结果显示 :成功地构建了 pZeoSV2 (+) /ANNⅡ真核表达质粒 ,经转染HL 60细胞后 ,可表达annexinⅡ活性蛋白 ,荧光检测显示其表达蛋白分布在转染细胞的膜表面 ;FCM检测annexinⅡ在转染后 48小时呈高表达 ;annexinⅡ可显著增加纤溶酶的活性 ,且瞬时表达及稳定表达具有相同的结果。annexinⅡ反义寡核苷酸与单抗均可显著抑制annexinⅡ的促纤溶活性 (P <0 .0 1 )。结论 :annexinⅡ在纤维蛋白溶解中具有重要作用 ,用本研究构建的annexinⅡ重组载体可为今后的相关研究提供实验基础 ,并为防治出血及血栓性疾病研究有可能提供新的思路和开辟新的途径。更多还原

【Abstract】 The study was designed to investigate annexinⅡ resulting in molecular pathological mechanism of the primary fibrinolysis and establish annexinⅡ vector model for further research on disturbance of coagulation. A target gene was amplified from human umbilical vein endothelial cells (HUVEC) by RT PCR. AnnexinⅡ gene fragment was purified and ligated with molecular biological recombinant technology. The recombinant of plasmid annexinⅡ was transfected into HL 60 cells and its distribution in the cell and structure characteristics of annexinⅡ protein was evaluated by multi photon excitation laser scanning microscope. By means of flow cytometry (FCM) and Werstern blot technique, the protein expression was qualitatively and quantitatively analyzed. Transfected cells were treated in vitro with annexinⅡ antisense oligonucleotide (AS) targeting to the start site of annexinⅡ cDNA. The results showed that the recombinant pZeoSV2(+)/ANNⅡ was constructed successfully and expressed in HL 60 cells. The protein expression was distributed on the surface of cell by fluorescence assay. After transfection for 48 hours, the cells occured higher level of expression. The level of the plasmin was significantly enhanced in the present of annexinⅡ. The FCM and Western blot analysis showed the annexinⅡ expression was similar both in transiently and stably transfected in HL 60 cells. AnnexinⅡ antisense oligonucletide and McAb significantly inhibited the activity of plasminogen. It was concluded that annexinⅡ plays an important role in the fibrinotysis. AnnexinⅡvector was defined as a expression tool for further studing fibrinolysis and coagulopathy in malignant disease.更多还原