流动状态下ADAMTS13羧基端的功能研究

发布时间：2018-01-01 18:41

本文关键词：流动状态下ADAMTS13羧基端的功能研究　出处：《华中科技大学学报(医学版)》2011年06期 　论文类型：期刊论文

【摘要】：目的研究ADAMTS13及其突变体于体外流动状态下裂解血管性血友病因子(VWF),降低血小板粘附形成血栓能力的差异。方法将全长ADAMTS13(FL)及缺失TSP2-8CUB1+2(MDTCS)和缺失CUB1+2(delCUB)cDNA质粒瞬时转染COS7细胞,Western blot检测各瞬时表达细胞分泌的蛋白。利用流体力学装置平板流动小室(FlowChamber)培养人脐静脉内皮细胞,并通过蠕动泵加压流动培养液模拟人体血管内流动状态,荧光显微镜观测加入AD-AMTS13或其突变体蛋白后荧光标记的血小板聚集状态的差异。结果成功在真核表达细胞内瞬时表达ADAMTS13及其突变体蛋白,简单纯化并经Western blot检测各表达蛋白相对分子量与预期一致。Flow Chamber模拟人体血管内剪切力作用下,FL蛋白和MDTCS蛋白可以不同程度地抑制血小板凝集,delCUB蛋白抑制血小板凝集的能力显著下降。结论成功瞬时表达全长ADAMTS13及其突变体蛋白。初步证实流动状态下ADAMTS13羧基端是参与裂解VWF以抑制血小板凝集的主要区域。
[Abstract]:Objective to study the effect of ADAMTS13 and its mutant on VWF in vitro. Methods to reduce the thrombotic ability of platelet adhesion. Methods the full-length ADAMTS13FLL and the deletion of TSP2-8CUB1 2 MDTCSs and CUB1 2 (2) were used to reduce the thrombotic ability of platelets. DelCUB)cDNA plasmid was transiently transfected into COS7 cells. Human umbilical vein endothelial cells (HUVECs) were cultured by Western blot. The flow state of human blood vessel was simulated by using peristaltic pump pressurized flow medium. Fluorescence microscope was used to observe the difference of platelet aggregation after adding AD-AMTS13 or its mutant protein. Results the transient expression of ADAMTS13 and its mutation in eukaryotic expression cells were successful. Body protein. Simple purification and detection of the relative molecular weight of the expressed proteins by Western blot. Flow Chamber simulated human intravascular shear stress. FL protein and MDTCS protein can inhibit platelet agglutination to some extent. Conclusion the transient expression of full-length ADAMTS13 and its mutant protein was successfully achieved. It was preliminarily confirmed that the carboxyl terminal of ADAMTS13 in mobile state is. Participate in the lysis of VWF to inhibit the main areas of platelet agglutination.
【作者单位】：华中科技大学同济医学院附属同济医院妇产科;郑州大学第一附属医院生殖中心;
【基金】：国家自然科学基金资助项目(No.30671093,No.30672243)
【分类号】：R554.6
【正文快照】： 血管性血友病因子裂解酶(A disintegrin andmetalloproteinase with thrombospondin motif 13,ADAMTS13,即von Willebrand factor-cleavingprotease,VWF-cp)是由Furlan[1]和Tsai[2]等在研究血管性血友病因子(von Willebrand factor,VWF)的生理特性时,从正常人血浆中纯化的一

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