蜈蚣提取液治疗肝癌Bel-7404细胞后的差异表达蛋白研究

Differentially expressed proteins in hepatocellular carcinoma cells treated with extract of centipede

目的筛选蜈蚣提取液(ECP)治疗肝癌Bel-7404细胞后的相关差异表达蛋白,探讨其药物治疗机制。方法一步抽提法制备肝癌Bel-7404细胞株治疗组ECP浓度(10 mg/mL)和对照组(RPIM1640完全培基培养)的总蛋白质,双向凝胶电泳建立两组总蛋白的双向凝胶电泳图谱,PDQuest软件对其图谱比较,MAL-DI-TOF-MS对差异表达的蛋白质进行分析并获取肽质量指纹图谱,数据库搜索鉴定主要差异表达蛋白质。结果共发现76个差异表达蛋白点(P<0.05)。治疗组中41个表达下调,35个表达上调。选择最明显的14个蛋白质斑点作质谱分析,其中肽基脯氨酰顺反异构酶A(PPIA)、核纤层蛋白-B1(Lamin-β1)、黄素还原酶(FRase)、转铁结合蛋白2(Tbp2)、唾液酸合成酶(SAS)、3-磷酸甘油醛脱氢酶(GAPDH)、周期素依赖激酶抑制蛋白1(P21)和半乳凝素(Gal-7)在治疗组中表达上调,角蛋白(CK-19)、肌动蛋白(Actin)、转内质网ATP(三磷酸腺苷酶)酶(TER ATPase)、ω-1谷胱甘肽转移酶(GSTO1-1)、抑制蛋白(PFN1)及碱性磷酸酶(ALP)在治疗组中表达下调。结论 ECP能诱导肝癌Bel-7404细胞内某些蛋白异常表达,能多途径抑制肝癌细胞的生长,诱导其凋亡或直接死亡。

【Objectives】 To screen the relevant differentially expressed proteins after treatment with extract of centipede（ECP）,and to investigate the molecule mechanism of ECP for HCC Bel-7404.【Method】 Firstly,comparative two-dimensional gel electrophoresis（2-DE） technology was adopted to separate the total protein of hepatocellular carcinoma（HCC） Bel-7404 treatment group by ECP and that of its control group respectively.The well-resolved,reproducible 2-DE patterns of treatment group of ECP and control group were established.Then,PDQuest software was used to analyze 2-DE images,and the differentially expressed proteins between the two groups were identified by both peptide mass fingerprint（PMF） and peptide sequence tag（PST） based on MALDI-TOF-MS（Matrix-assisted laser desorption/ionization time of flight mass spectrometry）.【Result】 We found out 76 protein spots at difference level of expression（P 0.05）,among which 41 spots decreasing and 35 spots increasing in treatment group.Among the identified 14 protein spots,the expression level of proteins which up-regulates in treatment group is 8 in total,including Peptidyl-prolyl cis-trans isomerase A（PPIA）,Lamin-β1,Flavin reductase（FRase）,Transferrin-binding protein 2（Tbp2）,Sialic acid synthase（SAS）,Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）,Cyclin dependent kinase proflin 1（P21） and Galectin-7（Gal-7）.But 6 proteins in treatment group is down-regulation,including Keratin（CK-19）,Actin,Transitional endoplasmic reticulum ATPase（TERATPase）,Glutathione transferase omega-1（GSTO1-1）,Profilin-1（PFN1） and Alkaline phosphatase（ALP）.【Conclusion】 ECP can change some proteins in HCC Bel-7404 to abnormal expression.The result hints that ECP can inhibit the growth of HCC Bel-7404 cell,and induce it to apoptosis or directly decease through multi-channels.