凝血因子ⅩⅢA基因Arg77Cys错义突变和Arg174stop无义突变致凝血因子ⅩⅢ缺陷的分子机制  

Molecular Mechanisms of Arg77Cys Missence Mutation and Arg174stop Nonsense Mutation of Factor ⅩⅢA Gene Causing Severe Factor ⅩⅢ Deficiency

作　　者：郑卫东[1,2] 刘艳辉[1,2] 罗盈[1,2] 姚志彬[3] 

机构地区：[1]广东省人民医院病理医学部检验科 [2]广东省医学科学院,广东广州510080 [3]中山大学中山医学院解剖和神经生物学系,广东广州510080

出　　处：《中国实验血液学杂志》2011年第6期1456-1461,共6页Journal of Experimental Hematology

摘　　要：本研究以凝血因子ⅩⅢ(FⅩⅢ)基因突变为研究的切入点,从分子水平研究这些突变致病的机制。构建野生型FⅩⅢA重组表达质粒,用定点突变方法获得含有2种突变(Arg77Cys及Arg174stop)的FⅩⅢA重组表达质粒。分别将上述重组质粒通过脂质体介导的基因转移方法转染到COS-7细胞表达;用发色底物法检测转染细胞中FⅩⅢ活性,荧光定量RT-PCR、Western blot及酶联免疫吸附实验(ELISA)检测转染细胞中人FⅩⅢA的RNA表达水平和蛋白表达量。结果表明:含有2种突变(Arg77Cys及Arg174stop)的FⅩⅢA重组质粒转染组FⅩⅢA mRNA相对表达丰度分别为0.82±0.21和0.76±0.17,与野生型FⅩⅢA重组质粒转染组(1.06±0.51)比较没有显著性差异(p>0.05)。野生型FⅩⅢA重组质粒转染细胞裂解物中FⅩⅢ活性为(61.6±30.4)%,浓缩的细胞培养上清液中FⅩⅢ活性为(24.0±2.9)%,而2种突变型FⅩⅢA重组质粒转染细胞裂解物及培养上清液FⅩⅢ活性明显减低。转染细胞裂解物的Western blot分析显示,含Arg77Cys错义突变的FⅩⅢA重组蛋白在培养细胞内含量明显减低,仅见1条微弱的条带。ELISA证实,野生型FⅩⅢA重组质粒转染细胞裂解物中FⅩⅢA∶Ag量为(32.8±14.5)%,浓缩的细胞培养上清液中FⅩⅢ∶Ag量为(13.2±2.3)%。而2种突变型FⅩⅢA重组质粒转染细胞裂解物及培养上清液中FⅩⅢA∶Ag水平极度减低,低于ELISA检测低限。结论:FⅩⅢA Arg77Cys错义突变和Arg174stop无义突变未导致FⅩⅢA mRNA水平的显著减低,但都可以导致FⅩⅢA蛋白水平的表达异常。在本研究先天性FⅩⅢ缺陷症患者中FⅩⅢA重度缺乏可能是FⅩⅢA Arg77Cys错义突变和Arg174stop无义突变的共同作用所致。The present study was aimed to investigate the molecular mechanisms responsible for the pathogenesis of severe factor ⅩⅢ（FⅩⅢ） deficiency.Site-directed mutagenesis was conducted to obtain human FⅩⅢA expression plasmids bearing the mutations.Wild type FⅩⅢA recombinant plasmid（pcDNA3.1-FⅩⅢA-wt） and 2 mutant FⅩⅢA recombinant plasmids（pcDNA3.1/FⅩⅢA/77mut,pcDNA3.1/FⅩⅢA/174mut） were transfected into the cultured COS-7 cells using lipofectamine 2000 transfection reagent,respectively.FⅩⅢ activities were measured by the Berichrom FⅩⅢ chromogenic assay.The expression levels of FⅩⅢA mRNA were detected by real-time RT-PCR.The recombinant FⅩⅢA mutants were determined by using Western blot and ELISA.The results showed that the normalized mRNA levels of 2 mutants in transfected COS-7 cells were 0.82±0.21 and 0.76±0.17,respectively.The relative levels of both mRNA transcripts were not significantly decreased as compared with the wild type（1.06±0.51）. FⅩⅢ activity and FⅩⅢA antigen levels in concentrated media of cell expressing the wild type protein were（24.0±2.9）% and（13.2±2.3）%,respectively.FⅩⅢ activity and FⅩⅢA antigen levels in cell lysates containing the wild type recombinant protein were（61.6±30.4）% and（32.8±14.5）%,respectively.However,the antigen levels and activity of 2 mutants were severely decreased as compared to the wild type.It is concluded that both mutations severely disturb the normal expression of FⅩⅢA protein.The reduction of expression levels and decreased activities of the 2 mutants provides a convincible explanation for the deficiency phenotype in the index case.

关 键 词：凝血因子ⅩⅢ缺陷症 Arg77Cys错义突变 Arg174stop无义突变 分子病理机制 

分 类 号：R554.5[医药卫生—血液循环系统疾病]