中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2015年
3期
212-217
,共6页
黄爱博%许华%洪文旭%庄志雄%刘建军
黃愛博%許華%洪文旭%莊誌雄%劉建軍
황애박%허화%홍문욱%장지웅%류건군
三氯乙烯%核蛋白质类%膜蛋白质类%人正常肝细胞系%RNA剪接
三氯乙烯%覈蛋白質類%膜蛋白質類%人正常肝細胞繫%RNA剪接
삼록을희%핵단백질류%막단백질류%인정상간세포계%RNA전접
Trichloroethylene%Nuclear proteins%Membrane proteins%Human hepatic L-02 cells%RNA splicing
目的 研究三氯乙烯(trichloroethylene)对人正常肝细胞系(L-02细胞)亚细胞蛋白质组的影响,探讨其潜在肝毒性作用机制.方法 采用0和8.0 mmol/L的三氯乙烯处理细胞后,分别提取三氯乙烯处理前后L-02细胞的细胞膜和细胞核总蛋白质,通过差异荧光双向凝胶电泳(2D-DIGE)筛选出差异点,运用基质辅助激光解析飞行时间串联质谱(MALDI-TOF-MS/MS)对差异蛋白点进行质谱鉴定.采用生物信息学方法对差异蛋白进行跨膜结构域(transmembrane domain)和基因本体(gene ontology,GO)功能聚类的分析,用Western blot分析验证不同浓度三氯乙烯处理下膜蛋白ATP合成酶β亚基(ATP5B)、核蛋白核不均一核糖核蛋白H2(hnRNP H2)和上游识别序列结合蛋白1(FUBP1)在L-02细胞中表达情况.结果 三氯乙烯作用于L-02细胞24 h后,鉴定出差异表达膜蛋白14个和差异表达核蛋白18个,在使用0、2.0、4.0和8.0 mmol/L的三氯乙烯处理细胞后,ATP5B蛋白相对表达量分别为1.00 ±0.03、1.21±0.14、1.25 ±0.12、1.48 ±0.17(F=8.51,P=0.007);hnRNP H2蛋白相对表达量分别为1.00 ±0.09、1.22±0.15、1.43 ±0.21、1.53±0.17(F =6.57,P=0.015);FUBP1蛋白相对表达量分别为1.00±0.11、0.91 ±0.07、0.73±0.04、0.67 ±0.03(F=15.81,P =0.001).生物信息分析显示,差异表达蛋白参与的生物学过程中,差异蛋白主要聚集在RNA加工(差异蛋白数为10个,P =2.46×10-6),特别是在RNA剪接上(差异蛋白数9个,P=1.77×10-7).结论 三氯乙烯处理可以引起亚细胞蛋白质组的改变,而这些与RNA剪接相关蛋白的异常表达为进一步研究三氯乙烯的肝毒性作用机制提供了新的线索.
目的 研究三氯乙烯(trichloroethylene)對人正常肝細胞繫(L-02細胞)亞細胞蛋白質組的影響,探討其潛在肝毒性作用機製.方法 採用0和8.0 mmol/L的三氯乙烯處理細胞後,分彆提取三氯乙烯處理前後L-02細胞的細胞膜和細胞覈總蛋白質,通過差異熒光雙嚮凝膠電泳(2D-DIGE)篩選齣差異點,運用基質輔助激光解析飛行時間串聯質譜(MALDI-TOF-MS/MS)對差異蛋白點進行質譜鑒定.採用生物信息學方法對差異蛋白進行跨膜結構域(transmembrane domain)和基因本體(gene ontology,GO)功能聚類的分析,用Western blot分析驗證不同濃度三氯乙烯處理下膜蛋白ATP閤成酶β亞基(ATP5B)、覈蛋白覈不均一覈糖覈蛋白H2(hnRNP H2)和上遊識彆序列結閤蛋白1(FUBP1)在L-02細胞中錶達情況.結果 三氯乙烯作用于L-02細胞24 h後,鑒定齣差異錶達膜蛋白14箇和差異錶達覈蛋白18箇,在使用0、2.0、4.0和8.0 mmol/L的三氯乙烯處理細胞後,ATP5B蛋白相對錶達量分彆為1.00 ±0.03、1.21±0.14、1.25 ±0.12、1.48 ±0.17(F=8.51,P=0.007);hnRNP H2蛋白相對錶達量分彆為1.00 ±0.09、1.22±0.15、1.43 ±0.21、1.53±0.17(F =6.57,P=0.015);FUBP1蛋白相對錶達量分彆為1.00±0.11、0.91 ±0.07、0.73±0.04、0.67 ±0.03(F=15.81,P =0.001).生物信息分析顯示,差異錶達蛋白參與的生物學過程中,差異蛋白主要聚集在RNA加工(差異蛋白數為10箇,P =2.46×10-6),特彆是在RNA剪接上(差異蛋白數9箇,P=1.77×10-7).結論 三氯乙烯處理可以引起亞細胞蛋白質組的改變,而這些與RNA剪接相關蛋白的異常錶達為進一步研究三氯乙烯的肝毒性作用機製提供瞭新的線索.
목적 연구삼록을희(trichloroethylene)대인정상간세포계(L-02세포)아세포단백질조적영향,탐토기잠재간독성작용궤제.방법 채용0화8.0 mmol/L적삼록을희처리세포후,분별제취삼록을희처리전후L-02세포적세포막화세포핵총단백질,통과차이형광쌍향응효전영(2D-DIGE)사선출차이점,운용기질보조격광해석비행시간천련질보(MALDI-TOF-MS/MS)대차이단백점진행질보감정.채용생물신식학방법대차이단백진행과막결구역(transmembrane domain)화기인본체(gene ontology,GO)공능취류적분석,용Western blot분석험증불동농도삼록을희처리하막단백ATP합성매β아기(ATP5B)、핵단백핵불균일핵당핵단백H2(hnRNP H2)화상유식별서렬결합단백1(FUBP1)재L-02세포중표체정황.결과 삼록을희작용우L-02세포24 h후,감정출차이표체막단백14개화차이표체핵단백18개,재사용0、2.0、4.0화8.0 mmol/L적삼록을희처리세포후,ATP5B단백상대표체량분별위1.00 ±0.03、1.21±0.14、1.25 ±0.12、1.48 ±0.17(F=8.51,P=0.007);hnRNP H2단백상대표체량분별위1.00 ±0.09、1.22±0.15、1.43 ±0.21、1.53±0.17(F =6.57,P=0.015);FUBP1단백상대표체량분별위1.00±0.11、0.91 ±0.07、0.73±0.04、0.67 ±0.03(F=15.81,P =0.001).생물신식분석현시,차이표체단백삼여적생물학과정중,차이단백주요취집재RNA가공(차이단백수위10개,P =2.46×10-6),특별시재RNA전접상(차이단백수9개,P=1.77×10-7).결론 삼록을희처리가이인기아세포단백질조적개변,이저사여RNA전접상관단백적이상표체위진일보연구삼록을희적간독성작용궤제제공료신적선색.
Objective To put the insight into the trichloroethylene (TCE)-induced effect on the differential expression of subcellular proteins in human normal liver cell line (L-02).Methods The membrane proteins and nuclear proteins of TCE-treated (8.0 mmol/L) group and controls were extracted by subcellular proteome extraction kit,respectively.The TCE-indueed differentially expressions were analyzed by a two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight spectrometry (MALDI-TOF-MS).Bioinformatics analysis was used to reveal the biological processes and predict transmembrane domains of differential expressed proteins.The expression of ATP synthase subunit beta (ATP5 B),heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) and far up steam element-binding protein 1 (FUBP1) were measured under TCE treatment by Western blot.Results After TCE treatment for 24 h in L-02 cells,14 membrane proteins and 18 nuclear proteins were identified as differential expression.After treated with TCE in concentrations of 0,2.0,4.0 and 8.0 mmol/L for 24 h,the relative levels of ATP5B expression were 1.00 ±0.03,1.21 ±0.14,1.25 ±0.12 and 1.48 ± 0.17 (F =8.51,P =0.007),the relative levels of hnRNP H2 expression were 1.00 ± 0.09,1.22 ± 0.15,1.43 ± 0.21,1.53 ± 0.17 (F =6.57,P =0.015),respectively;the relative levels of FUBP1 expression were 1.00 ±0.11,0.91 ±0.07,0.73 ±0.04 and 0.67 ±0.03 (F=15.81,P =0.001),respectively,which were consistent with the results in proteomics.The bioinformatics analysis showed that the most dominant biological process were involved in RNA processing (10 proteins,P =2.46 × 10-6),especially in RNA splicing (9 proteins,P =1.77 × 10-7).Conclusion The exposure of TCE could alter the expression of membrane proteins and nuclear proteins in L-02 cells.These abnormal expressed proteins involved in RNA splicing would provide novel clues for further understanding of TCE-induced hepatotoxicity.
