1例血小板无力症的分子发病机制研究  

Study on molecular pathogenesis for a case of Glanzmann′s thrombasthenia

作　　者：高丙晶 龚岩[1] 屈晨雪[1] 由然[1] 苗林子[1] 陆遥[1] 李涛[1] GAO Bingjing;GONG Yan;QU Chenxue;YOU Ran;MIAO Linzi;LU Yao;LI Tao(Department of Clinical Laboratory,Peking University First Hospital,Beijing 100034,China)

机构地区：[1]北京大学第一医院检验科

出　　处：《临床检验杂志》2019年第9期680-685,共6页Chinese Journal of Clinical Laboratory Science

摘　　要：目的探讨1例血小板无力症患儿的发病原因,阐明其致病机制。方法收集1例血小板无力症患儿外周血,通过基因测序技术明确致病基因位点;采用PCR定点突变方法制备突变质粒,转染中国仓鼠卵巢上皮细胞(CHO-K1),构建体外真核表达系统。用免疫印迹法检测突变型CHO-K1细胞中血小板αⅡb和β3蛋白的合成;流式细胞术检测突变型CHO-K1细胞膜和胞质αⅡb和β3表达水平;免疫荧光显微镜观察突变型CHO-K1中αⅡb和β3的表达及分布。结果该患儿为Ⅱ型血小板无力症。基因测序发现ITGB3基因存在2个突变,且尚未被报道。ITGB3 c.1495 T>C错义突变导致β3蛋白亚基第499号半胱氨酸被精氨酸代替(p.C499R);ITGB3 c.1728 delC移码突变导致β3蛋白亚基第577号丝氨酸开始的氨基酸合成发生改变,并在改变之后的第92个氨基酸终止。免疫印迹法结果为突变型CHO-K1细胞裂解液中均检测到αⅡb和β3一级结构的合成表达。流式细胞术检测结果为突变型CHO-K1细胞表面及胞内β3表达量缺如;荧光显微镜检测结果为突变型CHO-K1细胞未见β3蛋白亚基的分布。结论ITGB3 c.1495 T>C和c.1728delC突变是该患儿的发病原因,这2个突变并未影响血小板膜β3蛋白亚基一级结构合成,但影响其高级结构的表达和形成。Objective To investigate the molecular pathogenesis for a patient with Glanzmann thrombasthenia(GT).Methods The peripheral blood of a patient with Glanzmann′s thrombasthenia was collected,and the genetic mutations were detected by gene sequencing technology.The mutant plasmids were prepared by PCR site-directed mutagenesis and transfected into CHO-K1 cells of Chinese hamster ovary to construct in vitro eukaryotic expression system.The expressions of αⅡb and β3 protein subunits in CHO-K1 cells were detected by western blot.The expression levels of αⅡb andβ3 in cellular membrane and cytoplasm of CHO-K1 cells were detected by flow cytometry.The expression and distribution ofαⅡb and β3 in CHO-K1 cells were observed by immunofluorescent labeling under microscope.Results This patient was diagnosed with typeⅡGT.Gene sequencing revealed two mutations in ITGB3 gene which has not been reported in the literature.ITGB3 c.1495 T>C missense mutation resulted in replacement of cysteine no.499 by arginine(p.C499R).ITGB3 c.1728 delC code shift mutation resulted in a change in the amino acid synthesis initiated by the β3 protein subunit serine no.577 and terminated by the 92nd amino acid following these changes.The results of western blot showed that the synthesis and expression of primary structures ofαⅡb and β3 were detectable in the lysates of mutant CHO-K1 cells.The results of flow cytometry showed that no expression of β3 on the surface and intracellular of mutant CHO-K1 cells was observed.Under fluorescence microscopy no distribution ofβ3 protein subunit was displayed in mutant CHO-K1 cells.Conclusion The mutation of ITGB3 c.1728 del C or ITGB3 c.1495 T>C should be relevant to the cause of GT in this patient.The mutation of ITGB3 c.1728 del C and ITGB3 c.1495 T>C seems not to affect the formation of the primary structure ofβ3 protein subunit,but did affect the formation of its high-level structure.

关 键 词：ITGA2B ITGB3 血小板无力症 基因突变 

分 类 号：R446[医药卫生—诊断学]