VWF前导肽对D1区VWF突变体的作用机制研究  

The Effect of VWF Propeptide on VWF Mutant in D1 Domain

作　　者：虞秀群 马珍妮[1] 凌婧[2] 赵赟霄[1] 殷杰[1] 余自强[1] 阮长耿[1] YU Xiu-Qun;MA Zhen-Ni;LING Jing;ZHAO Yun-Xiao;YIN Jie;YU Zi-Qiang;RUAN Chang-Geng(National Clinical Research Center for Hematologic Diseases,Jiangsu Institute of Hematology,The First Affiliated Hospital of Soochow University,Key Laboratory of Thrombosis and Hemostasis of Ministry of Health,Suzhou 215000,Jiangsu Province,China;Children's Hospital of Soochow University,Department of Hematology and Oncology,Suzhou 215000,Jiangsu Province,China)

机构地区：[1]国家血液系统疾病临床医学研究中心,江苏省血液研究所,苏州大学附属第一医院,国家卫生健康委员会血栓与止血重点实验室,江苏苏州215000 [2]苏州大学附属儿童医院血液肿瘤科,江苏苏州215000

出　　处：《中国实验血液学杂志》2022年第5期1541-1548,共8页Journal of Experimental Hematology

基　　金：国家自然科学基金(81600105,81600106);江苏省自然科学基金(BK20180053)。

摘　　要：目的:探讨野生型VWFpp与前导肽D1区VWF突变体共转染是否能纠正D1区突变引起的VWF缺陷。方法:将4个定点诱变的VWF D1区突变体质粒单独瞬时转染HEK 293细胞;VWF D1区突变体质粒与VWFpp质粒共同瞬时转染HEK 293细胞,通过ELISA、垂直多聚体电泳分析细胞上清液及裂解液中的VWF,免疫荧光共聚焦显微镜下观察细胞内质网中VWF的滞留。结果:在垂直多聚体分析中,VWF突变体和VWFpp共表达后VWF多聚体条带消失。共表达后,无论是细胞上清液还是裂解液中VWF抗原含量均减少。在免疫荧光共聚焦显微镜下,共表达后,虽然细胞内VWF含量减少,但VWF在内质网滞留比率下降。结论:VWFpp虽然能减少D1区VWF突变体在内质网的滞留,促进了VWF在亚细胞器间的转运,但进一步抑制了D1区突变体VWF的合成及分泌。Objective: To investigate whether co-transfection of wild-type VWFpp with VWF mutant in D1 region is able to correct VWF defects in biosynthesis and secretion. Methods: Four VWF mutant plasmids were single transfected into HEK 293 cells, or co-transfected into HEK 293 cells with the wild type VWFpp plasmids. The VWF in supernatant and lysate of transfected cells were analyzed by ELISA, vertical VWF multimer electrophoresis. The retention of VWF in endoplasmic reticulum of transfected cells were detected by immunofluorescence confocal microscope. Results: In the vertical VWF multimer analysis, with co-expressing VWF mutant and VWFpp, the VWF multimer bands disappeared,and the VWF antigen in both supernatant and lysate of cells decreased, compared with the single expression of VWF mutant. Although the intracellular levels of VWF antigens decreased after co-expression, the retention rate of VWF mutant decreased in endoplasmic reticulum. Conclusion: VWFpp can reduce the retention of VWF in endoplasmic reticulum, assists the transport of VWF between subcellular organelles. However, VWFpp inhibits the biosynthesis and secretion of VWF about the mutant in D1 domain.

关 键 词：血管性血友病因子 血管性血友病因子前导肽 突变 生物合成 分泌 

分 类 号：R554.1[医药卫生—血液循环系统疾病]