中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2011年
10期
737-741
,共5页
周进%方丽%姚文秀%赵新%魏阳%周行%谢华%王理扬%陈俐娟
週進%方麗%姚文秀%趙新%魏暘%週行%謝華%王理颺%陳俐娟
주진%방려%요문수%조신%위양%주행%사화%왕리양%진리연
细胞培养氨基酸稳定同位素标记-质谱技术%槲皮素%HepG2细胞%热休克蛋白
細胞培養氨基痠穩定同位素標記-質譜技術%槲皮素%HepG2細胞%熱休剋蛋白
세포배양안기산은정동위소표기-질보기술%곡피소%HepG2세포%열휴극단백
Stable isotope labeling with amino acids in cell culture-mass spectrometry%Quercetin%HepG2 cells%Heat shock proteins
目的 观察槲皮素对人肝癌细胞HepG2中热休克蛋白(HSP)表达的影响,为其抗肿瘤活性探寻可能的靶点.方法 经过连续传代,充分氘化HepG2细胞中的亮氨酸,并鉴定其标记率.采用四甲基偶氮唑蓝(MTT)法检测槲皮素对HepG2细胞生长的抑制活性及有效浓度.以50 μmol/L槲皮素作用氘化HepG2细胞48 h后,与正常HepG2细胞按1:1等量混合电泳,提肽后进行质谱鉴定,将获得的肽片段数据进行Mascot检索.设定严格的可信区间,得出槲皮素作用前后HSP的定量信息.采用逆转录聚合酶链反应(RT-PCR)验证槲皮素作用前后HepG2细胞中HSP90和HSP70的表达差异.结果 连续传代10次以上,HepG2细胞中氘化亮氨酸已达95％以上.MT法检测结果表明,槲皮素对HepG2细胞的IC50接近50 μmol/L,且随着时间和浓度的增加,其对HepG2细胞的生长抑制作用更加明显.细胞培养氨基酸稳定同位素标记-质谱技术(SILAC-MS)获得了槲皮素作用前后HepG2细胞中HSP90、HSP70、HSP60和HSP20的定量信息.相对正常HepG2细胞而言,所有鉴定的HSP在50 μmol/L槲皮素作用48h后均有较大程度的表达下调,其中HSP90表达下调至正常HepG2细胞的49.3％,HSP70表达下调至正常HepG2细胞的43.6％.RT-PCR的检测结果支持SILAC-MS的鉴定结果.结论 SILAC-MS成熟可靠,能全面定量分析外因作用前后HepG2细胞中全系列蛋白谱的变化.槲皮素对HepG2细胞中HSP表现出强烈的抑制能力,并且这种作用可能是其发挥抗肿瘤活性的途径之一.
目的 觀察槲皮素對人肝癌細胞HepG2中熱休剋蛋白(HSP)錶達的影響,為其抗腫瘤活性探尋可能的靶點.方法 經過連續傳代,充分氘化HepG2細胞中的亮氨痠,併鑒定其標記率.採用四甲基偶氮唑藍(MTT)法檢測槲皮素對HepG2細胞生長的抑製活性及有效濃度.以50 μmol/L槲皮素作用氘化HepG2細胞48 h後,與正常HepG2細胞按1:1等量混閤電泳,提肽後進行質譜鑒定,將穫得的肽片段數據進行Mascot檢索.設定嚴格的可信區間,得齣槲皮素作用前後HSP的定量信息.採用逆轉錄聚閤酶鏈反應(RT-PCR)驗證槲皮素作用前後HepG2細胞中HSP90和HSP70的錶達差異.結果 連續傳代10次以上,HepG2細胞中氘化亮氨痠已達95％以上.MT法檢測結果錶明,槲皮素對HepG2細胞的IC50接近50 μmol/L,且隨著時間和濃度的增加,其對HepG2細胞的生長抑製作用更加明顯.細胞培養氨基痠穩定同位素標記-質譜技術(SILAC-MS)穫得瞭槲皮素作用前後HepG2細胞中HSP90、HSP70、HSP60和HSP20的定量信息.相對正常HepG2細胞而言,所有鑒定的HSP在50 μmol/L槲皮素作用48h後均有較大程度的錶達下調,其中HSP90錶達下調至正常HepG2細胞的49.3％,HSP70錶達下調至正常HepG2細胞的43.6％.RT-PCR的檢測結果支持SILAC-MS的鑒定結果.結論 SILAC-MS成熟可靠,能全麵定量分析外因作用前後HepG2細胞中全繫列蛋白譜的變化.槲皮素對HepG2細胞中HSP錶現齣彊烈的抑製能力,併且這種作用可能是其髮揮抗腫瘤活性的途徑之一.
목적 관찰곡피소대인간암세포HepG2중열휴극단백(HSP)표체적영향,위기항종류활성탐심가능적파점.방법 경과련속전대,충분도화HepG2세포중적량안산,병감정기표기솔.채용사갑기우담서람(MTT)법검측곡피소대HepG2세포생장적억제활성급유효농도.이50 μmol/L곡피소작용도화HepG2세포48 h후,여정상HepG2세포안1:1등량혼합전영,제태후진행질보감정,장획득적태편단수거진행Mascot검색.설정엄격적가신구간,득출곡피소작용전후HSP적정량신식.채용역전록취합매련반응(RT-PCR)험증곡피소작용전후HepG2세포중HSP90화HSP70적표체차이.결과 련속전대10차이상,HepG2세포중도화량안산이체95％이상.MT법검측결과표명,곡피소대HepG2세포적IC50접근50 μmol/L,차수착시간화농도적증가,기대HepG2세포적생장억제작용경가명현.세포배양안기산은정동위소표기-질보기술(SILAC-MS)획득료곡피소작용전후HepG2세포중HSP90、HSP70、HSP60화HSP20적정량신식.상대정상HepG2세포이언,소유감정적HSP재50 μmol/L곡피소작용48h후균유교대정도적표체하조,기중HSP90표체하조지정상HepG2세포적49.3％,HSP70표체하조지정상HepG2세포적43.6％.RT-PCR적검측결과지지SILAC-MS적감정결과.결론 SILAC-MS성숙가고,능전면정량분석외인작용전후HepG2세포중전계렬단백보적변화.곡피소대HepG2세포중HSP표현출강렬적억제능력,병차저충작용가능시기발휘항종류활성적도경지일.
Objective To detect the changes of heat shock protein(HSP)expression in human hepatocellular carcinoma HepG2 cells after treated by quercetin through a proteomics strategy termed SILAC (stable isotope labeling by amino acids in cell culture)-MS(mass spectrometry).Methods HepG2 cells cultured in d3-labeled DMEM medium were passaged for more than ten generations to reach an enough high labeling ratio.MTT assay was used to assess the inhibitory effect of quercetin on proliferation of HepG2 cells.In SILAC,total protein was extracted from control HepG2 cells and those treated by 50 μmol/L quercetin for 48 h,and then mixed to a 1:1 ratio.After in-gel digestion and idenfication by LC-MS/MS analysis,quantification informations of changed proteins were acquired by searching on Mascot 2.0 program (MatrixScience Ltd.,London)against SWISS-PROT protein database.To ensure a high confidence level for identification,those peptides with Mascot scores below the threshold value were excluded from analysis and not included in the list of quantified proteins(P ＜0.01).Protein abundance was calculated as ratios of the peak intensity of the fragment ions from the labeled versus the unlabeled peptides.RT-PCR was uesd to verify the reliability of HSPs changes by quercetin treatment from the SILAC-MS results.Results After passaged for ten generations,the d3-labeling ratio was above 95％.MTT showed that quercetin inhibited the proliferation of HepG2 cells obviously,with a IC50 close to 50 μmol/L,and in a dose-dependent and timedependent manner.The MS showed that the expression of almost all heat shock family proteins was downregulated a lot.The expression of HSP90 exposed to quercetin for 48 h was decreased to 49.3％ of the normal HepG2 cells,and the expression of HSP70 was decreased to 43.6％ of the normal Hep G2 cells.Quantitation information showed that the expression of HSP90α,HSP76,HSP60 and HSP27 was declined to 59.3％,44.2％,51.3％ and 62.6％,respectively.Those results demonstrated that the quantification for changed protiens by SILAC-MS was correct.Conclusions Quercetin can exert a significant inhibitory effect on whole expression of heat shock proteins in HepG2 cells.We suppose this maybe one of the pathways through which quercetin plays an important anti-tumor role.SILAC-MS is a reliale technique and can be used to quantify the changes of whole protein spectrum in HepG2 cells before and after treatment with some exogeneous factors.
