蛋白C P275S突变导致遗传性蛋白C缺陷症的分子机制研究

目的研究蛋白C（PC）基因P275S突变导致遗传性PC缺陷症的分子机制。方法构建PC基因野生型和P275S突变型表达质粒,并瞬时转染HEK293T细胞和COS 7细胞进行体外表达。ELISA检测转染细胞上清液和细胞裂解液的PC抗原;实时荧光RT-PCR（real time,RT-PCR）检测转染细胞PC mRNA表达量的改变;细胞免疫荧光染色检测PC在内质网和高尔基体内的分布。结果转染细胞上清液和细胞裂解液中的PC P275S抗原分别为野生型PC抗原的22.6%和78.9%;实时RT-PCR结果显示,与野生型PC相比,PC P275S突变体在mRNA水平没有减少;细胞免疫荧光染色显示,野生型PC蛋白在内质网和高尔基体中均有大量分布,而PC P275S突变蛋白主要位于内质网中。结论分泌障碍和细胞内降解是PC P275S导致PC缺陷症的原因。

Molecular Mechanisms of Protein C Deficiency Caused by P275S Mutation

Objective To study the molecular mechanisms of protein C（PC） deficiency caused by PC P275S mutation.Methods Wild-type and P275S mutant PC cDNA expression plasmids were constructed and transfected into HEK 293T cells and COS 7 cells,respectively.ELISA was used to detect the PC antigens.The expression of PC mRNA was investigated by real time RT-PCR.Immunofluorescent assay was utilized to analyze the distribution of PC in the endoplasmic reticulum and Golgi complex.Results Compared to the wide type,P275S mutant PC antigens in the supernatant of culture medium and cell lysates were 22.6% and 78.9% separately.Real time RT-PCR analysis of the total mRNA from transfected cells showed no reduction of the P275S mutant PC mRNA expression.Immunofluorescent assay revealed that wild type PC protein was distributed largely in both endoplasmic reticulum and Golgi complex,while the PC P275S mutant protein was mainly located in the endoplasmic reticulum.Conclusion Impaired secretion and degradation intracellularly of the mutant PC might be the molecular mechanisms of PC deficiency caused by P275S mutation.