紫杉醇耐药卵巢癌细胞亚株的蛋白组学研究

目的: 建立卵巢癌细胞株与紫杉醇耐药卵巢癌细胞亚株的差异蛋白表达图谱,寻找对紫杉醇药物耐受的相关蛋白。方法: 以人卵巢浆液性囊腺癌细胞株(SKOV3)和对紫杉醇稳定耐药卵巢浆液性囊腺癌细胞亚株(SKOV3-TR30)为研究对象,应用双向电泳技术建立SKOV3与SKOV3-TR30的蛋白质差异表达图谱,并对图谱进行质谱分析。然后应用蛋白免疫印迹和免疫细胞化学方法对其中3个有差异的蛋白进行定量和定位检测。结果: (1)经过组内与组间3次重复,发现每次实验均有相似变化的差异点23个。其中与SKOV3细胞株相比,在SKOV3-TR30细胞株表达上调大于3倍的蛋白质点有16个(P<0.05),表达下调大于3倍的蛋白质点有7个(P<0.05)。(2)通过质谱分析和数据库搜索,其中7个蛋白点得到初步鉴定,分别为可溶性抗药性相关钙结合蛋白(Sorcin)、组织蛋白酶B前体(Cathepsin B)、调宁蛋白3、热休克蛋白27(HSP27)、波形纤维蛋白、尼克酰胺乙酰乙酸水解酶和血红蛋白β链。(3)蛋白免疫印迹结果显示,Sorcin、Cathepsin B及HSP27在SKOV3-TR30细胞中的表达显著高于SKOV3细胞株(P<0.05)。免疫细胞化学的结果表明,SKOV3及SKOV3-TR30均可检测到Sorcin、Cathepsin B及HSP27阳性表达的细胞,并且3个蛋白均分布在细胞浆内,以核周最为明显。结论: 低浓度紫杉醇诱导的上皮性卵巢癌耐药细胞亚株中存在细胞骨架相关信号转导分子表达上调,上调的细胞骨架相关信号转导分子在卵巢癌临床预示耐药与逆转耐药治疗中的作用值得进一步研究。

Proteomics study on paclitaxel-resistant ovarian cancer subline

AIM: To assess the differential protein expression profiles of paclitaxel-resistant ovarian cancer sub-line and ovarian cancer cell line in order to detect the proteins associated with paclitaxel-resistance evolution.METHODS: The epithelial ovarian cancer cell line SKOV3 and its paclitaxel-resistant sub-line SKOV3-TR30 were selected. Total proteins from the two cell lines were separated by 2-DE and the differentially expressed proteins were identified by mass spectrometry. Then three protein (Sorcin,Cathepsin B,HSP27) expressions in paclitaxel- resistant cells were compared with parental cell line by Westerrn blot analysis and immunocytochemistry.RESULTS: (1) Twenty-three proteins spots showed significant changes in SKOV3-TR30 compared to SKOV3. Among them, sixteen proteins were found to be up-regulated and seven proteins were down-regulated in chemoresistant cells (expression regulation more than 3-fold). (2) Seven of these proteins were identified by tandem mass spectrometry as follows: sorcin,cathepsin B precursor,calponin-3,heat-shock protein 27,vimentin,nicotinate-nucleotide pyrophosphoylase,and hemoglobin beta chain. (3) Western-blot analysis indicated that sorcin, cathepsin B and HSP27 were up-regulated in SKOV3-TR30. Immunocytochemistry revealed that the three proteins were all located in cytoplasm, especially perinudear.CONCLUSION: The expression of signal transducing molecules associated with cytoskeleton were moderately up-regulated in paclitaxel-resistant ovarian cancer subline. These proteins may provide new targets for therapy, but their role in drug resistance in ovarian cancer, especially in vivo need a further study.