摘要
目的筛选慢性乙型肝炎患者经异甘草酸镁(MgIG)治疗前后血浆外泌体的差异蛋白质组。方法分别采集36例MgIG治疗慢性乙型肝炎患者前后的血浆标本(2 ml/例),利用超速离心方法提取血浆外泌体,利用Nanosight NS300颗粒粒度分析仪检测外泌体颗粒的浓度和内径。随机各抽取3例MgIG治疗前后组的血浆外泌体,裂解后提取蛋白,利用胰蛋白酶消化后,用液相色谱串联质谱技术进行label-free差异蛋白质组学分析,筛选出变化倍数1.5倍以上的差异蛋白。利用酶联免疫吸附测定技术对感兴趣差异蛋白的表达量进行验证(n=30)。计量资料的比较采用配对样本均数的t检验。结果提取的外泌体平均颗粒浓度为2.2×10^(9)个/ml,平均粒径为(107±52)nm,与理论血浆外泌体大小值相符,证明成功提取了血浆外泌体。蛋白质组学结果显示,MgIG治疗慢性乙型肝炎患者前后相比较,共筛选出153个差异蛋白,包括85个上调蛋白和68个下调蛋白。酶联免疫吸附实验结果显示,慢性乙型肝炎患者MgIG治疗后组和治疗前组相比较,血浆外泌体中肝细胞生长因子激活剂和肝细胞生长因子样蛋白浓度差异有统计学意义(P<0.05)。肝细胞生长因子激活剂在MgIG治疗前组血浆外泌体中浓度为(45.9±9.4)μg/ml,在MgIG治疗后组为(13.9±2.0)μg/ml。肝细胞生长因子样蛋白在MgIG治疗前组血浆外泌体中浓度为(23.4±4.9)μg/ml,在MgIG治疗后组为(13.8±2.2)μg/ml。酶联免疫吸附实验结果均与蛋白质组学结果一致。结论本研究成功筛选出MgIG治疗慢性乙型肝炎患者前后血浆外泌体差异蛋白质组,为研究MgIG治疗慢性乙型肝炎的分子机制提供实验依据。
Objective To screen the differential proteomic of plasma exosomes before and after magnesium isoglycyrrhizinate(MgIG)treatment in chronic hepatitis B patients.Methods Plasma samples were collected from 36 cases with chronic hepatitis B before and after MgIG treatment(2 ml/case).Plasma exosomes were extracted by ultracentrifugation.Exosomal particles concentration and inner diameter were detected by Nanosight NS300 particle size analyzer.Three cases of plasma exosomes were randomly selected beforec and after MgIG reatment.Proteins were extracted ater lysis and digsted with typsin.Label-frcce dfrential proteomis analysis was perfomed by liquid chromatogaplytandem mass spectrometry to screen out dferential proteins that changed more than 1.5 timnes.Enzyme linked immwunosorbent assy(ELISA)was used to verifly the quntiative difirenial proein epessos(n=30).Measurement data were compared by paired sample tHest.Results The average particle concentration of the extracted exosomes was 2.2×10^(9)/ml,and the avenage size was(107±52)nm,which was consistent with the theoretical value of plasma ecxosome sixe,proving that the plasma exosomes were sucssaiy extracted.Proteomics resuts showed that before and after MgIG treatment in chronic hepatis B patients,a total of 153 difreriallyl expressed proteins were screened,including 85 up-rcgulated and 68 down-regulated proteins.Enzyme-linked immunosorbent assay resuts showed that compared with the MgIG before and after treatment group of chronice hepatitis B patients,the dfferences in the concentrations of hepatocyte growth factor activator and bepatocyte growth factor like protein in plasma exosomes were stistically significant(P<0.05).Hepatocyte growth factor activator concentation in the plasma exosomes before and afler MgIG teatmeat group was(45.9±9.4)μg/m and(13.9±20)μg/ml,rspectively,and it was dowr-regulated by about 3 times.Hepatocyte growth fator-like protin concntation in the plasm exosomes before and afer MgIG treatment group was(23.4±4.9)μg/ml and(13.8±2.2)μg/ml,respetively,and it was down-regulated by about 2 times.Enzyme linked immunsorbent assy results had consistency with the protomis rsults.Conclusion This study scessfully screned the diferenial protcomie of plasma exosomes before and after MgG teatment in chronice hepatis B,and provided experimental basis for studyig the molecular mechanism ofMgIG teatment for chronic hepati B.
作者
肖二辉
胡水旺
宁会彬
康月花
殷辉
毛重山
康谊
尚佳
Xiao Erhui;Hu Shuiwang;Ning Huibin;Kang Yuehua;Yin Hui;Mao Zhongshan;Kang Yi;Shang Jia(Department of Infectious Diseases,Henan Provincial People Hospital,People's Hospital of Zhengzhou University,People's Hospital of Henan University,Zhengzhou 450003,China;Department of Pathophysiology,Southern Medical University,Guangdong Provincial Key Laboratory of Proteomics,Guangzhou 510515,China)
出处
《中华肝脏病杂志》
CSCD
北大核心
2021年第3期246-252,共7页
Chinese Journal of Hepatology
基金
中国肝炎防治基金会天晴肝病研究基金(20170121)
河南省医学科技攻关计划项目(201702204)
广东省医学科学技术研究基金(A2017577)。
