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 before and after MgIG treatment. Proteins were extracted after lysis and digested with trypsin. Label-free differential proteomics analysis was performed by liquid chromatography-tandem mass spectrometry to screen out differential proteins that changed more than 1.5 times. Enzyme linked immunosorbent assay (ELISA) was used to verify the quantitative differential protein expression (n = 30). Measurement data were compared by paired sample t-test. Results: The average particle concentration of the extracted exosomes was 2.2×10(9)/ml, and the average size was (107 ± 52) nm, which was consistent with the theoretical value of plasma exosome size, proving that the plasma exosomes were successfully extracted. Proteomics results showed that before and after MgIG treatment in chronic hepatitis B patients, a total of 153 differentially expressed proteins were screened, including 85 up-regulated and 68 down-regulated proteins. Enzyme-linked immunosorbent assay results showed that compared with the MgIG before and after treatment group of chronic hepatitis B patients, the differences in the concentrations of hepatocyte growth factor activator and hepatocyte growth factor like protein in plasma exosomes were statistically significant (P < 0.05). Hepatocyte growth factor activator concentration in the plasma exosomes before and after MgIG treatment group was (45.9 ± 9.4) μg/ml and (13.9 ± 2.0) μg/ml, respectively, and it was down-regulated by about 3 times. Hepatocyte growth factor-like protein concentration in the plasma exosomes before and after MgIG treatment group was (23.4 ± 4.9) μg/ml and (13.8 ± 2.2) μg/ml, respectively, and it was down-regulated by about 2 times. Enzyme-linked immunosorbent assay results had consistency with the proteomics results. Conclusion: This study successfully screened the differential proteomic of plasma exosomes before and after MgIG treatment in chronic hepatitis B, and provided experimental basis for studying the molecular mechanism of MgIG treatment for chronic hepatitis B.
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。.
Keywords: Chronic hepatitis B; Exosome; Magnesium isoglycyrrhizinate; Plasma; Proteomics.