p.Gly86Asp变异导致遗传性蛋白C缺陷症的致病机制分析  

Analysis of the molecular pathogenesis of hereditary protein C deficiency due to a p.Gly86Asp variant of the PROC gene

作　　者：蒋淑婷 王欢欢[1] 刘媚娜[1] 杨丽红[1] 金艳慧[1] 谢海啸[1] 徐琦煜[1] 王明山[1] Jiang Shuting;Wang Huanhuan;Liu Meina;Yang Lihong;Jin Yanhui;Xie Haixiao;Xu Qiyu;Wang Mingshan(Department of Clinical Laboratory,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou,Zhejiang 325015,China)

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江325015

出　　处：《中华医学遗传学杂志》2022年第7期685-688,共4页Chinese Journal of Medical Genetics

基　　金：浙江省科技厅公益技术研究计划(LGF18H080003);温州市公益科技项目(Y2020110)。

摘　　要：目的通过体外表达实验分析p.Gly86Asp变异导致遗传性蛋白C(protein C,PC)缺陷症的分子致病机制。方法构建野生型和p.Gly86Asp变异型PC表达质粒,分别转染HEK 293FT细胞。提取转染细胞内的总RNA,将RNA逆转录成cDNA,通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)法检测转染细胞内PROC基因的转录水平。收集细胞培养上清液和细胞裂解液,通过酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测PC抗原(PC antigen,PC:Ag)含量;采用蛋白免疫印迹试验(Western blotting,WB)检测PC含量以及相对分子质量。结果qRT-PCR结果显示野生型PC和p.Gly86Asp变异型PC在转录水平没有明显差异。与野生型PC:Ag含量相比,变异型PC:Ag含量在细胞培养上清液和细胞裂解液中分别为81.3%±2.6%和110.0%±2.8%。WB结果表明,野生型PC和变异型PC的相对分子质量没有明显差异;变异型PC的含量在细胞裂解液中明显高于野生型PC,而在细胞培养上清液中明显低于野生型PC。结论PC分泌障碍可能是p.Gly86Asp变异导致遗传性PC缺陷症的分子致病机制。Objective To explore the molecular pathogenesis of hereditary protein C(PC)deficiency due to a p.Gly86Asp variant of the PROC gene through in vitro expression experiment.Methods Wild type and Gly86Asp mutant expression plasmids of PC were constructed and respectively transfected into HEK 293FT cells.Total RNA was extracted from the transfected cells,and the expression of PROC gene was determined by quantitative real-time PCR(qRT-PCR).PC antigen(PC:Ag)in the supernatant of cell culture and cell lysate was determined by enzyme-linked immunosorbent assay(ELISA),and the level of PC protein was detected by Western blotting.Results qRT-PCR has detected no significant difference in the transcription level of wild-type and mutant-type PC.Compared with the wild type,the level of mutant PC:Ag in the supernatant and cell lysate were 81.3%±2.6%and 110.0%±2.8%,respectively.No difference was detected in the molecular weight between the wild-type and mutant-type PC by Western blotting.The PC content of mutant type was higher than wild-type in cell lysate,while the opposite was found with the cell culture supernatant.Conclusion The impaired secretion by mutant PC may be the molecular mechanism of PC deficiency caused by the p.

关 键 词：蛋白C缺陷症 PROC基因 基因变异 体外表达实验 

分 类 号：R596[医药卫生—内科学]