摘要
目的观察槲皮素对人肝癌细胞HepG2中热休克蛋白（HSP）发达的影响，为其抗肿瘤活性探寻可能的靶点。方法经过连续传代，充分氚化HepG2细胞中的亮氨酸，并鉴定其标记率。采用四早基偶氮唑蓝（MTT）法检测槲皮素对HepG2细胞中的亮氨酸,并鉴定其标记率。采用四早基偶氮唑蓝（MTT）法检测槲素对HepG2细胞生长的抑制活性及有效浓度。以50μmol/L槲皮素作用氚化HepG2细胞45h后，与正常HepG2细胞按1：1等量混合电泳，提肽后时行质谱鉴定，将获得的肽片段数据进行Mascot检索。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肿瘤活性的途径之一。
Objective To detect the changes of heat shock protein （lISP） expression in human hepatocellular carcinoma HepG2 cells after treated by quercetin through a proteomics strategy termed SII,AC （stable isotope labeling by amino acids in cell culture ） -MS （mass spectrometry）. Methods ttepG2 cells cultured in d3-1abeled DMEM medium were passaged fbr more than ten generations to reach an enough high labeling ratio. MTI＇ assay was used to assess the inhibitory effect of quercetin on prolit＇eration of HepG2 cells. In SII,AC, 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 anti idenfication by I,C-MS/MS analysis, quantification informations of changed proteins were acquired by searching on Mascot 2.0 prngram （ MatrixScience Ltd. , l,ondon） 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 ibr ten generations, the d3-1abeling ratio was above 95%. MTT showed that quercetin inhibited the proliferation of HepG2 cells obviously, with a ICso 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.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2011年第10期737-741,共5页
Chinese Journal of Oncology
