【作者】 白雪；

【导师】 李世拥；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 【目的】结直肠癌肝转移是影响结直肠癌预后的重要因素,25%的结直肠癌患者确诊时即伴随肝转移,另有25%患者在结直肠癌根治术后伴随肝转移。结直肠癌肝转移的早期诊断治疗对延长患者生命至关重要。为了深入研究结直肠癌发生、发展及肝转移的分子机理,以提高对结直肠癌肝转移的防治效果,我们采用蛋白质组学、细胞生物学及免疫组织化学研究方法,对结直肠癌原发灶和肝转移灶及不同分期的结直肠癌组织进行实验研究,为揭示结直肠癌肝转移机制及筛选临床肿瘤标志物和治疗靶标提供依据。【材料和方法】标本采集实验所用标本均取自北京军区总医院普通外科,选取经临床影像学诊断为结直肠癌伴随肝转移患者共16例,术中切取适量的结直肠癌原发灶、肝转移灶和癌旁正常肠粘膜标本,立即贮于液氮中冻存备用。所选标本术后病理证实为结直肠中低分化腺癌并伴随肝转移,临床TNM分期Ⅳ期(T2-4N1-2M1)。其中男9例女7例。年龄41-75岁。其中结肠癌8例,腹膜反折外直肠癌8例。蛋白质样品制备及双向凝胶电泳双向荧光差异凝胶电泳(2-D DIGE)按照GE Healthcare提供的Ettan DIGE使用手册操作方法进行。将每例患者的各种样品等量混和作为内标(Cy2标记),然后将各样品50μg分别与CyDye DIGE最小标记法染料(Cy3、Cy5)400pmol混和上样。将样品加入IPG胶条槽内并放置好24cmIPG胶条。使用等电聚焦仪PROTEAN IEF Cell ( Bio-Rad )进行第一向等电聚焦电泳(IEF);使用Ettan DALT Twelve电泳系统(Amersham)进行蛋白样品的二向分离。电泳结束后室温下在避光容器中将凝胶存放于SDS电泳缓冲液中,立即对凝胶进行扫描。图像采集与分析用Typhoon 9410扫描仪在488/520nm, 532/580nm, 633/670nm波长分别对Cy2, Cy3, Cy5荧光染料标记的图像进行扫描,用DeCyder v.5.02图像分析软件对DIGE图像进行分析和差异点寻找。计算各块胶与MASTER胶的匹配率,差异点满足条件:p≤0.05。质谱分析及质谱数据库搜寻使用液相色谱-电喷雾-串联质谱(LC-ESI-MS/MS)及MALDI-TOF-MS质谱仪对胶上消化后的差异蛋白点进行质谱分析。获得的肽序列信息(PSD)及肽质量指纹谱(PMF)数据用Mascot软件在SWISSPORT数据库中搜寻鉴定蛋白质。细胞生物学实验用常规RT-PCR方法克隆差异表达蛋白基因全长cDNA。构建真核表达载体pcDNA3.1-CAII。采用脂质体转染法将pcDNA3.1-CAII转染人直肠腺癌HR-8348细胞。四唑盐(MTT)比色试验:以转染pcDNA3.1-CAII的细胞为实验组;以未转染pcDNA3.1-CAII的细胞为对照组。分别观察5-Fu及奥沙利铂药物对其的抑制率。抑制率=[1-(实验组吸光度值/空白对照组吸光度值)]×100%。免疫组织化学实验用常规免疫组织化学方法验证人精氨酸酶在结直肠癌不同分期各种组织中的表达情况。使用美国ZYMED公司SP试剂盒在组织石蜡切片上进行免疫染色。【结果】双向凝胶电泳及质谱鉴定在16个病例组结直肠癌原发灶、肝转移灶及正常肠粘膜组织标本的2-D DIGE中,蛋白点清晰可辨,每块胶获得蛋白点约为900个。结直肠癌原发灶与肝转移灶蛋白质组分有明显差别。用DeCyder v.5.02图像分析软件对DIGE图像进行分析和差异点寻找。我们选择了22个差异蛋白点进行了质谱鉴定。鉴定出有意义的蛋白质20种。2种蛋白在各种癌组织中低表达而在正常肠粘膜中高表达,分别为碳酸酐酶II(CAII)和蛋白质二硫键异构酶(PDI);2种蛋白在结直肠癌原发灶组织中表达上调而在肝转移灶中表达下调,分别为激活蛋白因子2B,腺苷蛋氨酸变异体;16种蛋白在肝转移灶组织中表达上调,包括锌指蛋白64同系物,鸟嘌呤核苷酸交换因子4,人精氨酸酶,人谷胱甘肽S-转移酶A3,肿瘤坏死因子α-诱导蛋白9,谷氨酸脱氢酶载脂蛋白链A,转接分子受体2,核苷酸还原酶M2多肽等。细胞生物学实验转染CAII基因的直肠腺癌细胞HR8348对奥沙利铂和5-Fu化疗药物更敏感,耐药性更低。免疫组化验证人精氨酸酶定位结直肠癌组织细胞质中,人精氨酸酶在正常肠粘膜、无淋巴结转移的结直肠癌原发灶组织、有淋巴结转移的直肠癌原发灶组织、有肝转移的直肠癌原发灶组织、肝转移灶组织中的阳性表达率逐步增高。特别是在伴淋巴结转移的结直肠癌和肝转移灶组织中的表达阳性率显著高于无淋巴结转移的结直肠癌原发灶组织及正常肠粘膜。【结论】应用差异蛋白质组学方法研究结直肠癌发生过程中蛋白质组的改变,,是筛选结直肠癌肿瘤标志物及探讨结直肠癌发生及转移机制的有效手段。双向荧光差异凝胶电泳(2-D DIGE)作为近几年广泛应用的蛋白质组学新技术也开始应用于结直肠癌的研究,其精确度和可重复性都大大超过常规方法。本实验应用双向荧光差异凝胶电泳方法,对结直肠癌原发灶、肝转移灶及癌旁正常肠粘膜三种组织进行差异蛋白质组学研究,发现碳酸酐酶II(CAII)、蛋白质二硫键异构酶(PDI)在癌组织和复发转移灶中表达显著下调;发现激活蛋白因子2B(activator protein 2B)和腺苷蛋氨酸变异体(transgelin variant)在结直肠癌原发灶组织中表达显著增高。发现人精氨酸酶(Homo sapiens arginase)、谷胱甘肽转移酶A3(GSTA3)、鸟嘌呤核苷酸交换因子4 (ASEF)等在肝转移灶组织中表达显著增高;转染CAII基因的直肠腺癌细胞HR8348增强了奥沙利铂和氟尿嘧啶(5-Fu)对该细胞的细胞毒作用,使癌细胞对化疗药物更敏感,耐药性更低,提示癌组织中CAⅡ表达与癌组织的侵袭、转移和生物学行为有关,CAⅡ在抑制结直肠癌发生、转移中发挥作用,为肿瘤的基因诊断及基因治疗奠定了理论基础。免疫组织化学方法检测结直肠癌不同阶段各种组织发现,人精氨酸酶在伴淋巴结转移的结直肠癌原发灶和肝转移灶组织中的表达阳性率显著高于无淋巴结转移的结直肠癌原发灶组织和正常肠粘膜组织。人精氨酸酶的高表达不仅与结直肠癌的发生相关,在结直肠癌的发展转移,特别是淋巴结转移、肝转移中发挥重要作用。人精氨酸酶可能成为结直肠癌淋巴结转移、肝转移、预后评估的候选蛋白标志物。我们对结直肠癌肝转移做了初步的差异蛋白质组学研究,获得了一些差异蛋白,这些蛋白将是我们进一步研究结直肠癌肝转移机理的突破口,更深入的研究这些蛋白质在肿瘤的发生及转移过程中的作用,对揭示肝转移发生的机制和发现新的有意义的肿瘤标志物有一定的意义。本研究发现人精氨酸酶可能成为预测结直肠癌肝转移的标志物,但尚需要大样本多中心的临床实验进一步验证。相信,随着结直肠癌肝转移蛋白质组学研究的深入,肝转移发生机制将进一步阐明,新的有意义的生物标志物将被发现。更多还原

【Abstract】 Colorectal cancer is one of the common malignant tumors in our country. A large number of statistical data indicates that the rates of incidence and mortality of the disease in our country have been going up in recent years.Liver metastasis caused severe and fatal effect on patients that underwent radical resecsion for large intestine primary cancer. Early forecast and diagnosis of liver metastasis is the key to improving survival rate for coloretcal cancer patients. Now proteome study methods and techniques, especially two-dimensional electrophoresis (2-DE), provide useful and important support for the research of liver metastasis of coloretcal cancer.【Objective】To study differential proteins and their biological functions associated with colorectal cancer genesis and liver metastasis by proteomics and molecular biology immunohistochemistry techniques.【Methods】The samples of colorectal cancer liver metastasis corresponding to sixteen patients were tested with proteomic analysis. And the highly sensitive two-dimensional differential gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) for the identification of proteins differentially expressed in primary cancer lesion and liver metastasis cancer of colorectal cancer were used. The differentially expressed proteins found by 2-D DIGE would be confirmed and validated by immunohistochemistry analyses in those few cases which antibodies were available. Transfection experiment of rectum cancer HR-8348 cells was performed with the differential protein cDNA, and the changes of cell biological behavior was observed.【Results】Significant differences of protein expression were found on two-dimensional electrophoresis. Twenty two differential protein spots were analysed and identified. Human carbonic anhydraseⅡand protein disulfide isomerase were detected in normal colorectal mucosa, but lost in primary cancer lesion. activator protein 2B and transgelin variant were expressed in primary cancer. The expressions of zinc finger protein 64 homolog, guanine nucleotide exchange factor 4, Homo sapiens arginase, homo sapiens glutathione S-transferase A3, tumor necrosis factor alpha-induced protein 9, chain A structure of human glutamate dehydrogenase-apo form, toll-like receptor adaptor molecule 2, ribonucleotide reductase M2 polypeptide increased in the liver metastasis lesion in comparison to those in the primary cancer lesion.After transfection with human carbonic anhydraseⅡcDNA, the HR-8348 cell changed obviously with reduce of drug tolerance. Homo sapiens arginase expressions was assayed by immunohistochemistry method in Colorectal carcinoma associated tissue.【Conclusion】Differential expression of proteins are found among the normal colorectal mucosa ,colorectal cancer genesis and liver metastasis. Loss of carbonic anhydrase II expression and protein disulfide isomerase and enhancement expression of activator protein 2B and transgelin variant are related with colorectal carcinogenesis and colorectal cancer cell biological behavior. Enhancement expression of arginase ,zinc finger protein 64 homolog, guanine nucleotide exchange factor 4 and are related with colorectal cancer liver metastasis. This study of the colorectal cancer proteome represents a step toward a better understanding of the mechanism of the colorectal cancer liver metastasis,and arginase perhaps could be used as biomarkers for colorectal cancer liver metastasis risk更多还原