同时放化疗敏感性不同中晚期宫颈癌蛋白质组学比较的初步研究

Preliminary comparative proteomics study of cervical carcinoma tissues with different sensitivity to concurrent chemoradiotherapy

目的比较同时放化疗高敏感和低敏感中晚期宫颈癌组织之间蛋白质组的差异，识别和鉴定高敏感组和低敏感组之间差异表达蛋白，为中晚期宫颈癌患者同时放化疗敏感性的预测提供依据。方法搜集治疗前的中晚期宫颈癌组织标本10例，病理诊断均为中分化鳞状细胞癌。在进行同时放化疗后，根据WHO实体瘤疗效判断标准将宫颈癌组织标本分为高敏感组（5例）和低敏感组（5例）。提取组织总蛋白，进行二维凝胶电泳得到凝胶图谱；采用PD-quest 7．0软件进行匹配和差异分析，识别两组之间表达差异蛋白点。将这些差异蛋白点进行胶内原位切割、酶解后进行MALDI-TOF-MS分析，获取肽质量指纹图，数据库搜索鉴定蛋白质。应用Western blot方法检测其中2个差异表达蛋白Galectin-7和热休克蛋白70在10例宫颈癌组织标本中的表达。应用免疫组织化学方法检测Galectin-7和热休克蛋白70在95例临床活检中的表达情况。结果建立了分辨率高、重复性好的宫颈癌同时放化疗敏感组和不敏感组的双向凝胶电泳图谱。高敏感组蛋白点数为（781±74）个，低敏感组蛋白点数为（766±52）个，组间平均匹配率为87．6％。选择部分差异在2倍以上的蛋白斑点进行质谱分析、19个差异表达蛋白被成功鉴定，其中8个蛋白质在高敏感组高表达，10个蛋白质在高敏感组低表达。免疫印迹和免疫组织化学结果显示：热休克蛋白70在低敏感组中的表达率显著高于高敏感组，而Galectin-7在高敏感组中的表达率显著高于低敏感组，这一结果也与蛋白质组筛选结果基本一致。结论同时放化疗高敏感组和低敏感组宫颈癌组织间存在蛋白表达的差异，这些差异表达的蛋白可能与放化疗敏感性有关。

Objective To investigate the proteomics differences between the high-sensitivity（HS） and the low-sensitivity （LS） groups of cervical carcinoma treated by concurrent chemoradiotherapy, and to confirm the sensitivity associated proteins in intermediate stage and advanced cervical carcinoma. Methods Fresh carcinoma tissues were collected from 10 untreated cervical carcinoma patients. According to the response to concurrent chemoradiotherapy ,the tissues were classified into HS group and LS group. In the first part of our experiment, protein separation was performed using two-dimensional gel electrophoresis （ 2-DE ） with Amersham 18 cm linear pH 3-10 immobilized pH gradient（IPG） strips. The images of the gels were analyzed by PD-quest 7.0 software to find the differentially expressed protein-spots in each group. Then the differentially expressed protein-spots were incised from the gels and digested by trypsin. The peptide mass fingerprintings （PMF） was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. The proteins were identified by data searched in the Mascot-database. Two differentially expressed proteins were assayed by western blot and immunohistochemical methods. Results Most of the gels were clear and successfully analyzed by PD-quest 7.0 software. Most of the protein-spots concentrated on the area of 20-100 KDa（Mw） and pH4-8. The average number of the protein-spots was 781±74 in HS group and 766±52 in LS group. The match rate was 87.6% between the two groups. Eight proteins highly in HS group but lowly expressed in LS group included hemoglobin subunit beta,caspase-14 precursor, calmodulin-like, S100-A9 protein（ MRP-14 ） , galectin-7, HSKERC4, keratin 19 and actin. Ten proteins highly in LS group but lowly expression in HS group included anti HBs antibody light-chain Fab, lamin- B1, WARS protein, flavin reductase, glutamate dehydrogenase 1, nuclear matrix protein 238, retinal dehydrogenase 1, AF165172, subunit of replicative DNA polymerase and HSP70. The higher expression of HSP70 in LS group and galectin7 in HS groups were further confirmed by western blot and immunohistochemical method, Conclusions The 2-DE gels images are successfully acquired from high-sensitivity group and low-sensitivity group of intermediate stage and advanced cervical carcinoma tissues treated by concurrent chemoradiotherapy. Some differentially expressed proteins between the two groups can be further confirmed by western blot and immunohistochemical method.