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目的研究抗凝血酶(AT)基因 T98I 和 A404T 突变致 AT 缺陷症的分子机制。方法构建 AT 野生型和突变体表达质粒(ATwt、AT T98I、AT A404T)并瞬时转染至 COS-7细胞或 CHO 细胞,进行体外表达试验、脉冲-追踪试验和细胞免疫荧光染色;用荧光实时 PCR(RT-PCR)检测转染细胞 AT mRNA 表达量的改变;蛋白降解抑制实验检测突变蛋白在细胞内的降解途径。结果 AT T98I未从转染细胞内分泌且在细胞内逐渐降解,AT A404T 仅部分从细胞内分泌、大部分未分泌并在细胞内逐渐降解。RT-PCR 显示,与野生型 AT mRNA 相比,突变体 AT mRNA 不降低。脉冲-追踪试验发现,这两种 AT 突变体分泌障碍,未在细胞内聚集而是降解。蛋白降解抑制实验显示,突变体 AT T98I通过蛋白体酶途径进行细胞内降解。转染细胞荧光染色显示,转染 AT T98I 质粒的 CHO 细胞内荧光强度明显减弱、无明显核周聚集,而转染 AT A404T 质粒的 CHO 细胞内荧光减弱且弥散分布于胞质、核周有轻度聚集。结论分泌障碍和细胞内降解是 AT 基因 T98I 和 A404T 突变导致 AT 缺陷症的分子病理机制。
Objective To study the molecular mechanism of AT deficiency caused by antithrombin (AT) gene T98I and A404T mutations. Methods AT wild-type and mutant expression plasmids (ATwt, AT T98I, AT A404T) were constructed and transiently transfected into COS-7 cells or CHO cells for in vitro expression, pulse-tracking and immunofluorescence staining. PCR (RT-PCR) was used to detect the expression of AT mRNA in transfected cells. The protein degradation inhibition assay was used to detect the degradation pathway of mutein in cells. Results AT T98I was not secreted from the transfected cells and gradually degraded in the cells. AT A404T was only partially secreted from the cells, and most of them were not secreted and gradually degraded in the cells. RT-PCR showed that mutant AT mRNA did not decrease compared to wild-type AT mRNA. Pulsed-chase experiments revealed that these two AT mutants secreted disorders that did not aggregate but degrade in the cells. Protein degradation inhibition experiments show that the mutant AT T98I undergoes intracellular degradation through the proteasome pathway. Fluorescence staining of transfected cells showed that the fluorescence intensity of CHO cells transfected with AT T98I plasmid was significantly weakened and no obvious perinuclear aggregation was found. However, the fluorescence of CHO cells transfected with AT A404T plasmid was weakened and dispersed in the cytoplasm, Degree of aggregation. Conclusions Secretion disorder and intracellular degradation are the molecular pathological mechanisms of AT gene T98I and A404T mutations leading to AT deficiency.