CAJ | 학술논문

目的应用比较蛋白质组学方法寻找HBV X蛋白(HBx)作用相关蛋白,揭示HBx在肝细胞痛发生、发展中作用的分子机制.方法以转染HBx基因的人肝癌细胞株HepG2-Px和转染空白载体的人肝癌细胞株HepG2-P.为研究对象,应用二维凝胶电泳技术分离2种细胞的总蛋白质,图像分析识别差异表达的蛋白质点,通过基质辅助激光解吸电离飞行时间质谱及电喷雾电离串联质谱对差异表达的蛋白质点进行鉴定,蛋白质免疫印迹法验证部分差异表达的蛋白质在2株细胞中的表达水平.数据比较采用t检验.结果得到分辨率较高、重复性较好的2株细胞系的二维凝胶电泳图谱,质谱分析共鉴定32个HBx作用相关蛋白,包括25个在HepG2-Px中表达上调的蛋白质,如热休克蛋白90AB1、Bcl-2相关抗原-2,细胞核磷酸化蛋白、细胞内氯离子通道-1、基质金属蛋白酶-3和黑色素瘤相关抗原-12等,7个表达下调的蛋白质,如Wnt-5a等.蛋白质印迹验证了部分差异表达蛋白在2株细胞中的表达水平.结论筛选出的差异表达蛋白主要涉及宿主细胞的基囚转录、信号转导、细胞生长和增殖、细胞周期、细胞凋亡、DNA修复和细胞代谢、免疫应答等过程.提示这些与HBx相互作用的蛋白质可能与肝细胞癌的发生、发展相关,为揭示HBx的作用机制提供了线索.
목적 응용비교단백질조학방법심조HBV X단백(HBx)작용상관단백,게시HBx재간세포통발생、발전중작용적분자궤제.방법 이전염HBx기인적인간암세포주HepG2-Px화전염공백재체적인간암세포주HepG2-P.위연구대상,응용이유응효전영기술분리2충세포적총단백질,도상분석식별차이표체적단백질점,통과기질보조격광해흡전리비행시간질보급전분무전리천련질보대차이표체적단백질점진행감정,단백질면역인적법험증부분차이표체적단백질재2주세포중적표체수평.수거비교채용t검험.결과 득도분변솔교고、중복성교호적2주세포계적이유응효전영도보,질보분석공감정32개HBx작용상관단백,포괄25개재HepG2-Px중표체상조적단백질,여열휴극단백90AB1、Bcl-2상관항원-2,세포핵린산화단백、세포내록리자통도-1、기질금속단백매-3화흑색소류상관항원-12등,7개표체하조적단백질,여Wnt-5a등.단백질인적험증료부분차이표체단백재2주세포중적표체수평.결론 사선출적차이표체단백주요섭급숙주세포적기수전록、신호전도、세포생장화증식、세포주기、세포조망、DNA수복화세포대사、면역응답등과정.제시저사여HBx상호작용적단백질가능여간세포암적발생、발전상관,위게시HBx적작용궤제제공료선색.
Objective To identify hepatitis B virus X-interactive proteins by comparative proteomics method and to understand the molecular mechanism of HBx in the development of hepatocellular carcinoma(HCC).Methods Two-dimensional gel electrophoresis(2-DE)was used to separate the total proteins of HBx-transfected human hepatoma cell lines HepG2-Px and its parental cell lines HepG2-P_0.PDQuest software was applied to analyze 2 DE images.The differentially expressed protein spots between the two cell lines were identified by matrix-assisted laser desorptionionization time of flight mass spectrometry(MALDI-TOF-MS)and electrospray ionization tandem mass spectrometry(ESI-Q-TOF).Then,the differential expression levels of some identified proteins were determined by Western blot.The data were compared using t test.Results The well-resolved,reproducible 2-DE patterns of HepG2-Px and HepG2-P_0 total proteins were established.A total of 32 differential proteins were identified in HepG2-Px cell,including 25 up-regulated proteins,such as heat shock protein(HSP)90AB1,Bcl-2 associated athanogene(BAG)-2,nucleophosmin(B23),chloride intracellular channel(CLIC)-1,matrix metalloproteinase(MMP)-3,melanoma antigen(MAGE)-12,and 7 down-regulated proteins,such as Wnt-5a.The differential expression levels of some proteins between the two cell lines were confirmed by Western blot analysis.Conclusions Most of the identified proteins are involved in many processes,such as transcription,signal transduction,cell proliferation,cell cycle regulator,apoptosis,DNA repair,metabolisms and immunity.These differential proteins may play a role in tumor genesis and HC development.The data are valuable for further study on the role of HBx in human hepatocellular carcinoma.