宫颈上皮内瘤变与正常宫颈组织差异表达蛋白分析

目的探讨蛋白质组学方法筛查正常宫颈与宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)组织中的差异表达蛋白质标志物,为研究CIN发生的分子机制及临床诊治工作提供依据。方法收集2008年8月至2009年9月间首都医科大学附属北京妇产医院妇瘤科收治的正常子宫颈组织因子宫肌瘤手术行全子宫切除术患者9例为对照组、CIN组织23例(其中CINⅠ7例、CINⅡ8例、CINⅢ8例)。应用二维荧光差异凝胶电泳(two-dimensional fluorescence difference in gel electrophoresis,2-DDIGE)及DeCyder软件寻找差异表达蛋白质点,基质辅助激光解析飞行时间串联质谱(MALDI-TOF/TOF MS)分析差异蛋白质点,数据库搜索鉴定差异表达最明显的S100蛋白家族A9(S100 calcium-binding protein A9,S100A9),真核延长因子1α1(eukaryoticelongation factor 1-alpha-1,eEF1A1)及丙酮酸激酶M2(pyruvate kinase M2,PKM2)3种蛋白质;应用免疫组织化学法(immunohistochemistry,IHC)(其中正常宫颈组织10例、CINⅠ10例、CINⅡ10例、CINⅢ10例)及蛋白印记法(Western blotting)(其中正常宫颈组织12例,CINⅠ～Ⅱ12例,CINⅢ12例)进一步验证上述3种蛋白在CIN组织和正常宫颈组织的表达差异。结果获得CIN组织与正常宫颈组织2-D DIGE图,成功鉴定25个蛋白;免疫组化及蛋白印迹验证结果提示:S100A9蛋白表达于细胞质中,在CIN中表达水平高于正常组,差异有统计学意义(P=0.010);eEF1A1蛋白表达于细胞质中、PKM2蛋白表达于细胞核中,2者在CIN中表达水平均低于正常组,差异有统计学意义(P分别为0.352,0.000)。结论在正常宫颈组织与CIN组织之间存在差异蛋白质表达,S100A9蛋白可能成为辅助诊断CIN的相关标志物,PKM2蛋白可能成为抑制CIN发生的蛋白质,并可根据2种差异蛋白表达预测CIN的发生发展。

Analysis of differentially expressed proteins of cervical intraepithelial neoplasia tissues and normal cervix

Objective To discuss protein markers expressed differentially in normal cervix and cervical intraepithelial neoplasia (CIN) tissues by means of proteomics, providing the new basis to CIN molecular pathogenesis study, clinical diagnosis and treatment. Methods Uterine cervical tissue specimens were collected from the patients between August 2008 and September 2009 in Department of Oncology of Beijing Obstetrics and Gynecology Hospital, from patients with CIN (n=23, CINⅠ 7, CINⅡ 8, CINⅢ 8) and normal cervix (n=9 as controls). Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to find the differentially expressed protein-spots. Then matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was used to analyze the differentially expressed proteins and further to validate 3 strikingly differential proteins S100 calcium-binding protein A9(S100A9), eukaryotic elongation factor 1-alpha-1(eEF1A1)and pyruvate kinase M2 (PKM2) by database searching. Immunohistochemistry (IHC)(n=40, normal cervix 10、CINⅠ 10、CINⅡ 10、CINⅢ 10) and Western blotting (WB)(n=36,normal cervix=12、CINⅠ~Ⅱ 12、CIN Ⅲ~12)were used to verify the differently expressed proteins of S100A9, PKM2 and eEF1A1 between normal cervix and CIN tissues. Results CIN and normal cervix spectrometry were obtained by 2-D DIGE; and Appraisal 25 proteins successfully. IHC and Western blotting test results indicated that S100A9 protein was expressed in the cytoplasm, the expression in CIN levels were higher than that in normal group (P=0.010 ); eEF1A1 protein was expressed in cytoplasm and PKM2 protein was expressed in the nucleus, both the expressions of protein in CIN were lower than that in normal group (P = 0.352 and 0.000). Conclusion There are different protein expressions between CIN and normal cervix tissues. S100A9 protein may be regarded as a cancer marker associated with CIN; PKM2 proteins may be regarded as tumor suppression protein of CIN and the contents of these two differential proteins may help predict the development of CIN.