摘要
目的 探讨糖蛋白 (GP)Ⅸ基因点突变G2 113→A在导致巨血小板综合征 (BSS)中的意义。方法 用限制性内切酶分析患者和其直系亲属以及 4 0名正常人等位基因 ;采用定点诱变技术构建含有GPⅨ点突变G2 113→A的质粒PD ⅨG2 113A ;用含有GPⅠbα ,GPⅠbβ和GPⅨ或GPⅨ突变型全长编码序列的质粒对中国仓鼠卵巢 (CHO)细胞共转染 ;流式细胞仪检测转染后的CHO细胞表面GP的表达 ;免疫染色和免疫印迹分析转染后的CHO细胞胞浆中GPⅠbα和GPⅨ的表达。结果 先证者为纯合子 ,母亲和兄长均为杂合子 ;突变型CHO细胞膜上GPⅨ和GPⅠbα的表达显著减少 ,但大量存在于细胞胞浆中。结论 本例BSS患者的发病机制为GPⅨAla139(GCC)→Thr(ACC)突变。该突变不影响GPⅠb Ⅸ在细胞内的合成与组装 ,但影响其在细胞膜表面的锚定与表达。
Objective To identify a mutation G2113→A in the glycoprotein (GP)Ⅸ gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis. Methods Allele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-ⅨG2113A harboring the mutation G2113→A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPⅠbα, GPⅠβ and GPⅨ or mutant GPⅨ, respectively. Expression of GPⅠbα and GPⅨ in transfected CHO cells were analysed with flow cytometer. GPⅠbα and GPⅨ in the cytoplasma of transfected CHO cells were analysed by immunostainning and Western blotting. Results The patient was found to be homozygosity of the subsitution, her mother and her brother be heterozygous. Expressions of GPⅠbα and GPⅨ in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma. Conclusion The mutation of Ala139(GCC)→Thr(ACC) in the GPⅨ did not affect synthesis and assembly of GPⅠb/Ⅸ complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2003年第9期480-483,共4页
Chinese Journal of Hematology
基金
国家自然科学基金资助项目 (3 9870 3 43 )
关键词
巨血小板综合征
糖蛋白
基因突变
限制性内切酶
Bernard-Soulier syndrome
Glycoprotein
Gene
Restriction enzyme
Site-directed mutagenesis
