抗凝血酶基因C2759T(Leu99Phe)突变导致抗凝血酶缺陷症的分子机制研究

目的研究抗凝血酶(AT)基因C2759T(Leu99Phe)突变引起AT缺陷症的分子机制。方法用大引物法构建C2759T突变体AT重组表达质粒(ATM2759)，并将其和正常AT重组质粒(ATN)分别用Superfect试剂转染COS7细胞或CHO细胞，进行体外表达试验和细胞免疫荧光染色。、结果转染ATM2759的COS7细胞，培养上清液和细胞裂解液中的AT抗原(AT：Ag)分别为ATN的35．63％和174．97％，而培养上清液中的AT活性(AT：A)为ATN的47．73％。细胞免疫荧光试验证实，转染ATM2759的CHO细胞，其荧光强度明显高于转染ATN的CHO细胞。结论Leu99Phe 突变可能不是由于影响AT与肝素的结合而导致AT缺陷，而是由于突变导致AT分泌障碍和细胞内滞留所致。

Study on the molecular mechanism of antithrombin gene C2759T (Leu99Phe) mutation causing antithrombin deficiency

OBJECTIVE: To study the molecular mechanism of antithrombin (AT) gene C2759T (Leu99Phe) mutation causing AT deficiency. METHODS: A mutated AT cDNA expression plasmid ATM2759 was constructed by megaprimer method. ATM2759 and wild type AT cDNA expression plasmid ATN were transfected into COS7 cells or CHO cells by using Superfect reagent respectively for in vitro expression study and immunofluorescence assay. RESULTS: The antigen levels of AT (AT:Ag) in the cell lysate of ATM2759 transfected COS7 cells and the cell culture supernatant were 174.97% and 35.63% of that of ATN transfected COS7 cells respectively, whereas the AT activity in the cell culture supernatant was 47.73% of the control's. Immunofluorescence analysis showed that the fluorescence intensity was significantly higher in ATM2759 transfected CHO cells than in those transfected with ATN. CONCLUSIONS: Leu99Phe substitution may not affect the binding capacity of AT with heparin. Secretion defect and intracellular accumulation of the mutated AT protein might be the mechanisms of this mutation causing AT deficiency.