同时放化疗敏感性不同宫颈癌组织的二维电泳图谱建立及质谱分析

Two-dimensional Gel Electrophoresis and Mass Spectrometry of cervical Carcinoma Tissues with Different Concurrent Chemoradiotherapy Sensitivity

目的:分析同时放化疗后高敏感和低敏感中晚期宫颈癌组织之间蛋白质组的差异,为确定宫颈癌同时放化疗敏感性相关蛋白提供依据。方法:收集治疗前的中晚期宫颈癌活检组织标本,均为中分化鳞癌,置于—80℃超低温冰箱保存。同时放化疗后,根据WHO实体瘤疗效判断标准,将收集的10例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,进行双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)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。将部分差异蛋白点进行胶内原位酶解后进行MALDI-TOF-MS分析,获取肽质量指纹图,数据库搜索鉴定蛋白质。结果:建立了分辨率高,重复性好的同时放化疗后宫颈癌组织高敏感组和低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%。质谱分析成功鉴定15个差异表达蛋白,其中7个蛋白质在高敏感组高表达,8个蛋白质在高敏感组低表达。结论:蛋白质2-DE图谱和质谱鉴定结果说明同时放化疗后高敏感组和低敏感组宫颈癌组织间存在蛋白质表达的差异,这些蛋白质可能与同时放化疗敏感性有关。

Objective： To investigate the proteomic differences between the high-sensitivity （HS） group and the low-sensitivity （LS） group of cervical carcinoma tissues in middle-later stage after being treated by concurrent chemoradiotherapy, and to provide experimental evidences for ascertaining the concurrent chemoradiotherapy sensitivity associated proteins. Methods： The fresh carcinoma tissues were collected from 10 untreated cervical carcinoma patients and preserved at -80℃. According to their response to concurrent chemoradiotherapy, the tissues were classified into two groups： high-sensitivity group （HS） and low-sensitivity group （LS）. Protein was separated by using two-dimensional gel electrophoresis （2-DE） with Amersham 18cm linear pH 3-10 immobilized pH gradient （IPG） strips. The images of the gels were acquired by the scanner and then analyzed by using PD-quest7.3 software to find the different expression protein-spots between the two groups. Then the differentially expressed protein-spots was excised from the gels and digested by trypsin. The peptide mass fingerprinfings （PMF） was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and the proteins were identified by data searching in the Mascot-database. Results： Most of the gels were clear and successfully analyzed by PD-quest 7.3 software. The average number of the protein-spots was 781 ± 74 in high-sensitivity group and 766± 52 in the low -sensitivity group. The match rate of proteins was 87.6% between two groups. 7 high expression proteins were found in HS group which were low expression in LS group, 8 high expression proteins were found in LS group which were low expression in HS group. Conclusions： The 2-DE gels image of HS group and LS group of cervical carcinoma tissues in middle-later stage treated by concurrent chemoradiotherapy were successfully acquired, There were some differences in protein expression between HS group and LS group, and these differentially expressed proteins may be associated with concurrent chemoradiotherapy sensitivity.