A non-invasive diagnostic approach is crucial for the evaluation of severity of liver disease,treatment decisions,and assessing drug efficacy.This study evaluated plasma proteomic profiling via an N-terminal isotope tagging strategy coupled with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry measurement to detect liver fibrosis staging.Pooled plasma from different liver fibrosis stages,which were assessed in advance by the current gold-standard of liver biopsy,was quantitatively analyzed.A total of 72 plasma proteins were found to be dysregulated during the fibrogenesis process,and this finding constituted a valuable candidate plasma biomarker bank for follow-up analysis.Validation results of fibronectin by Western blotting reconfirmed the mass-based data.Ingenuity Pathways Analysis showed four types of metabolic networks for the functional effect of liver fibrosis disease in chronic hepatitis B patients.Consequently,quantitative proteomics via the N-terminal acetyl isotope labeling technique provides an effective and useful tool for screening plasma candidate biomarkers for liver fibrosis.We quantitatively monitored the fibrogenesis process in CHB patients.We discovered many new valuable candidate biomarkers for the diagnosis of liver fibrosis and also partly identified the mechanism involved in liver fibrosis disease.These results provide a clearer understanding of liver fibrosis pathophysiology and will also hopefully lead to improvement of clinical diagnosis and treatment.
LI ShuLongLIU XinWEI LaiWANG HuiFenZHANG JiYangWEI HanDongQIAN XiaoHongJIANG YingHE FuChu
目的为了探讨乙型肝炎病毒(HBV)X基因与慢性乙型肝炎及肝癌发生的关系,构建HBV X基因(hep-atitis B virus X gene,HBx)真核表达载体及其肝癌(HCC)细胞系HepG2瞬转模型,并检测HBx对HepG2细胞表型的影响。方法以pCMV-HBx为模板,PCR法扩增HBx基因编码区全长序列,将其克隆至pcDNA3.1-Flag真核表达载体中,构建重组真核表达载体pcDNA3.1-Flag-HBx,转染至HepG2细胞;Western印迹法检测细胞中HBx蛋白的表达水平;5-溴尿苷(5-溴脱氧尿嘧啶核苷,5-bromo-2'-deoxyuridine,BrdU)标记法检测转染HBx后细胞DNA合成变化;细胞周期检测试剂盒(CycleTESTTMPLUS DNA Reagent Kit)检测转染HBx后细胞周期的变化;AnnexinⅤ-APC/7-AAD法检测转染HBx后细胞凋亡情况。结果重组真核表达载体pcDNA3.1-Flag-HBx经双酶切和测序证明构建正确,转染该载体的HepG2细胞可检测到HBx蛋白的表达;与转染空质粒pcDNA3.1-Flag的细胞相比,细胞增殖能力增强,而细胞凋亡比率下降。结论成功构建了HBx基因真核表达载体,并研究了HBx对细胞表型的影响,为进一步探索HBx参与HBV引发HCC的分子机制奠定了基础。
