遗传性凝血因子XIII缺陷症分子机制的研究

目的 研究两种遗传性凝血因子(FXIII)A基因的缺陷(FXIIIA Arg77→Cys,Ser413→Trp)以了解其致病的分子机制。方法 构建正常人FXIIIA重组表达质粒(wt-PCI／FXIIIA),通过定点突变获得上述2种突变的FXIIIA重组表达质粒(mut-PCI／FXIIIA),并分别将它们转染到COS7细胞表达,PCR、RT-PCR和Western印迹检测转染细胞中人FXIIIA的DNA水平、RNA水平及其蛋白质的表达量。用脉冲追踪试验追踪人FXIIIA蛋白在细胞内的变化。通过生物素化戊胺的掺入检测细胞内、外人FXIIIA的活性。结果 wt-PCI／FXIIIA和mut-PCI／FXIIIA稳定转染的COS7细胞内FXIIIA在RNA水平表达量相同;而两种mut-PCI／FXIIIA转染的细胞内未检测到人FXIIIA蛋白质,且蛋白活性Ser413→Trp突变者仅为正常的8．7％,Arg77→Cys突变者则为0％。用脉冲追踪法显示正常FXIIIA蛋白质各时间点含量无减少,而两种突变的人FXIIIA在0．5h和1h虽存在,但很快消失。结论 两种FXIIIlA基因突变导致FXIIIA在细胞内不稳定,迅速被降解,从而FXIIIA蛋白量减少和活性丧失。

Molecular mechanisms of two novel mutations of factor XIII gene resulting in hereditary coagulation deficiency

OBJECTIVE: To investigate the mechanisms of two novel missense mutations of factor XIIIA subunit gene (Arg77-->Cys,Ser413-->Trp) in the pathogenesis of hereditary factor XIII deficiency. METHODS: Site-directed mutagenesis was conducted to obtain 2 mutant human XIII A recombinant plasmids, mut-PCI/FXIIIA. Normal wild type factor XIII A recombinant plasmid, wt-PCI/FXIIIA, and mut-PCI/FXIIIA, were transfected into cultured COS7 cells line, renal fibroid cell of African green monkey using Superfect reagent respectively, The expression levels of DNA, RNA and protein of human factor XIII, both wild type and mutant, were detected by PCR, RT-PCR and Western blotting. Pulse-chase experiment was used to look into the changing of factor XIII A in the cytoplasm. Factor XIIIA activity was assayed by Biotin-pentylamine incorporation technique. RESULTS: The mRNA levels of the two mutants in transfected cells were similar to that of the wild type factor XIIIA. But the amount of mutant factor XIIIA protein and its activity in cells decreased markedly, even disappeared. Pulse-chase experiment revealed that at the two mutants existed chase time 0.5 h and 1 h considerable amounts in cells and then disappeared rapidly later. CONCLUSION: The 2 mutations of the factor XIIIA cause the instability, degradation, and rapid disappearance of FXIIIA in cytoplasm, thus resulting in hereditary factor XIII deficiency.