胆管癌的蛋白质组学分析

背景与目的:胆管癌是目前常见的消化道恶性肿瘤,早期诊断困难,总体5年生存率极低,所以改善预后的关键在于早期诊断.近年来,蛋白质组学的迅猛发展,为检测肿瘤标志物提供了一种新技术.本研究通过比较胆管癌组织与正常胆管组织的蛋白质组表达差异,寻找胆管癌相关的蛋白质,选择敏感的分子标志物.方法:运用蛋白质组学技术,对16例胆管癌患者的胆管癌组织和正常胆管组织进行胶内差异双向凝胶电泳(2-DE),选择差异表达超过2倍的蛋白质进行MALDI-TOF质谱分析和生物信息学分析.结果:成功建立胆管癌组织和正常组织的双向凝胶电泳图谱,胆管癌组织和正常胆管组织平均蛋白质斑点数分别为1 087和1 048,其中表达差异超过2倍的斑点共有32个,质谱分析和数据库检索共鉴定出18种蛋白质,包括衔接蛋白成纤维细胞生长因子受体底物2(fibroblast growth factor recptor substrate 2,FRS2)、连环蛋白(catenin,beta 1)、细胞外信号调节激酶(extracellular response kinase)等.从功能上分析,这些差异蛋白质与癌细胞的发生、增殖、分化、转移等相关.结论:胆管癌组织与正常胆管组织间有18种差异蛋白质可能为研究胆管癌的生物学行为提供新的分子标记物.     

胆管癌组织及胆汁中端粒酶活性的检测及意义

目的：检测胆管癌组织和胆汁中的端粒酶活性，以研究与端粒酶与胆管癌的关系及对胆管癌的诊断意义。方法：用PCR—ELISA方法检测20例胆管癌的癌组织和胆汁中端粒酶活性，同时用相同的方法检测20例正常胆管组织和胆汁中的端粒酶活性以作对照。结果：20例胆管癌组织中，端粒酶活性阳性为80％（16／20），胆汁中端粒酶活性阳性率75％（15／20）。正常胆管组织端粒酶活性阳性表达率为0（0／20），胆汁中端粒酶活性阳性率为0（0／20）。结论：端粒酶可能参与了胆管癌的发生发展过程，检测胆汁中端粒酶活性可有助于胆管癌的诊断。     

蛋白质组学技术与肿瘤标志物研究

肿瘤标志物一般指肿瘤组织及细胞所产生的异常表达的活性物质包括激素、酶、特异性和非特异性的糖蛋白或糖脂等 ,但随肿瘤分子生物学研究的进展 ,肿瘤标志物的范围进一步扩展。肿瘤标志物最初的研究可追溯到 1 848年 ,英国医生 Bencejones发现骨髓瘤患者尿中的一种球蛋白可以作为诊断此病的标志 ,后来将此蛋白称为本琼氏蛋白以纪念此人的贡献。但此后很长一段时间 ,肿瘤标志物研究几乎处于停滞的阶段。直到 1 975年单克隆抗体技术问世之后 ,研究者得以从肿瘤抗原的角度用微量抗原制备大量的抗体来寻找特异性的标志物。后来采用免疫检测技术…     

胆汁中生物标志物检测对胆管癌诊断价值的研究进展

胆管癌是一种起源于胆管上皮细胞的恶性肿瘤,发病率逐年上升,预后不良.缺乏敏感、特异的早期诊断标志物和替代治疗是患者生存受限的主要原因.虽然血液中的生物标志物更常用于区分疾病和健康状况,但血液中大量混杂因素的干扰限制了其在胆管癌诊断中的应用.鉴于胆汁中富含与胆管上皮细胞代谢途径相关的代谢物,正在成为特别针对胆管癌的生物标...     

蛋白质组学在肺癌诊治研究中的应用

随着人类基因组计划的完成,双向凝胶电泳和质谱的应用以及生物信息学的引入,蛋白质组学的研究获得了极大发展.肿瘤蛋白质组学从新的角度研究肿瘤,寻找肿瘤标志蛋白,进而阐明其表达水平的变化与肿瘤发生、发展的相互关系,对肿瘤的早期诊断、治疗、药物研发等方面具有重要意义.本文就蛋白质组学的相关技术、策略及其在肺癌研究中的应用作一简要综述.     

中枢神经系统血管母细胞瘤的蛋白质组学分析

背景与目的:中枢神经系统血管母细胞(centralnervoussystemhemangioblastoma,CNSHB)的治疗较难,至今病因不明。本研究应用蛋白质组学方法分析HB与正常脑组织的蛋白质表达谱的差异,分离鉴定与HB发病的蛋白质,为研究HB的组织学起源与发病机制提供依据。方法:收集5例血管母细胞瘤与5例正常脑组织标本。提取标本的总蛋白质。通过双向凝胶电泳(2-DE)分离蛋白,确定血管母细胞瘤与正常脑组织的差异表达蛋白质点。应用基质辅助激光解离-飞行时间质谱(MALDI-TOF-MS)鉴定并对鉴定蛋白质进行生物信息学分析。结果:通过双向凝胶电泳,建立了约含600个蛋白质点的血管母细胞瘤及正常脑组织的高分辨率蛋白质表达图谱。两者相比具有较高的同源性。应用ImageMaster2D图像分析软件共发现115个差异蛋白质点。经MALDI-TOF-MS分析后成功鉴定了87个蛋白质点共计47种蛋白质。其中HB组较正常对照组表达上调46个蛋白质点,下调41个蛋白质点。鉴定蛋白质的功能涉及转运、蛋白质折叠与代谢、三羧酸循环、细胞分化、干细胞相关蛋白质等数个方面。对鉴定所得蛋白质Vimentin、14-3-3蛋白进行免疫组化染色可重复本研究的结果。结论:中枢神经系统血管母细胞瘤可能是一种起源于间叶脑组织的肿瘤。其发生是一个多因子参与、多步骤的复杂过程。多种蛋白质如Vimentin及14-3-3蛋白参与了血管母细胞瘤的发病并起到重要的作用。     

骨肉瘤与正常骨组织的蛋白质组学分析

目的 比较骨肉瘤与正常骨组织蛋白质组学的差异,探寻骨肉瘤特异性标志物。方法分别提取5例骨肉瘤肿瘤标本和5例正常骨组织总蛋白,进行双向电泳,重复3次;银染、扫描后进行ImageMaster 2-DE Platinum 6.0软件分析以发现差异蛋白并对差异蛋白质点进行MALDI -TOF/TOF质谱鉴定,获取肽质指纹图谱后应用NCBI数据库查找匹配蛋白质。结果获得重复性较好的双向电泳银染图谱,骨肉瘤组织蛋白质点数约为(1 296±179),正常骨组织蛋白质点数约为(1 108±162),明显差异的蛋白质点25个,其中10个被成功鉴定,6个蛋白质在骨肉瘤中表达上调,以谷胱甘肽转移酶升高最为突出;4个蛋白质在骨肉瘤中表达下降,以核苷二磷酸激酶下降最明显。结论蛋白质组学的分析能成功发现骨肉瘤与正常骨组织之间的差异蛋白质,为寻找骨肉瘤特异标志物提供依据。     

骨肉瘤与正常骨组织的蛋白质组学分析

目的 比较骨肉瘤与正常骨组织蛋白质组学的差异,探寻骨肉瘤特异性标志物.方法 分别提取5例骨肉瘤肿瘤标本和5例正常骨组织总蛋白,进行双向电泳,重复3次;银染、扫描后进行ImageMaster 2-DE Platinum 6.0软件分析以发现差异蛋白并对差异蛋白质点进行MALDI-TOE TOF质谱鉴定,获取肽质指纹图谱后应用NCBI数据库查找匹配蛋白质.结果 获得重复性较好的双向电泳银染图谱,骨肉瘤组织蛋白质点数约为(1 296±179),正常骨组织蛋白质点数约为(1108±162),明显差异的蛋白质点25个,其中10个被成功鉴定,6个蛋白质在骨肉瘤中表达上调,以谷胱甘肽转移酶升高最为突出;4个蛋白质在骨肉瘤中表达下降,以核苷二磷酸激酶下降最明显.结论 蛋白质组学的分析能成功发现骨肉瘤与正常骨组织之间的差异蛋白质,为寻找骨肉瘤特异标志物提供依据.     

人肺腺癌A549及A549/DDP细胞株差异蛋白质组学分析

 目的 比较人肺腺癌细胞株A549和耐顺铂株A549/DDP细胞的蛋白表达谱,筛选肺癌耐药相关蛋白,为 阐明肿瘤细胞的耐药提供新线索。 方法 对两种细胞株进行比较蛋白质组学研究,利用双向凝胶电泳技术（2-DE）分离细胞总蛋白,经 图像分析软件分析、基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）鉴定差异蛋白质点 ,并用免疫细胞化学（ICC）对部分差异蛋白进行验证。 结果 获得分辨率高、重复性好 的A549、A549/DDP总蛋白图谱,初步筛选出差异蛋白质点,经质谱分析及数据库检索,鉴定出 13种差异表达蛋白质,这些蛋白与细胞代谢、结构、解毒和DAN修复、以及信号转导等相关。 结论 A549、A549/DDP细胞株存在差异表达蛋白质点,提示这些蛋白可能与A549细胞耐顺铂相关,为 研究肺癌耐药提供依据。     

鼻咽癌淋巴结转移相关分子蛋白质组学技术筛选与鉴定

目的：比较2组不同程度颈部淋巴节结转移的初诊鼻咽癌组织在蛋白质组水平上的表达差异。方法：将52例2010-06-01—2011-03-31中山大学肿瘤防治中心初诊鼻咽癌患者,按照颈部淋巴结转移程度分为A组（N0～N1）和B组（N2～N3）,分别收集每组患者的肿瘤组织,提取总蛋白,进行双向凝胶电泳分离和质谱分析,利用UMAX Power Look 1100透射扫描仪获取凝胶图像,应用PDQuest 7．1．0软件包进行图像分析,选取凝胶图谱中差异表达〉1．5倍且P〈0,01的蛋白质斑点,应用基质辅助激光解吸／电离-飞行时间质谱仪（matrix assisted laser desorption ionization time of flight／mass spectrometry,MALDI-TOF／MS）进行蛋白质的肽指纹图谱鉴定。结果：在2组颈部淋巴节结转移程度不同的初诊鼻咽癌患者肿瘤组织间,共有7个蛋白质点强度差异≥1．5倍且P〈0．01。经质谱分析鉴定发现,B组中的表达量比在A组中的表达量,Stathmin升高〉5倍,缺氧诱导因子1-“抑制剂升高〉10倍。结论：Stathmin和缺氧诱导因子1-α抑制剂在高淋巴结转移的鼻咽癌组织中表达增加,提示其在肿瘤发生发展机制中的作用可能与淋巴结转移有关。     

胆管恶性肿瘤患者胆汁细菌学分析

目的:探讨胆管癌胆汁中的细菌分布及特征。方法:对西安交通大学医学院第二、第三附属医院2011年11月至2012年10月期间诊断为胆管癌患者的胆汁进行细菌培养并分析。结果:共106例患者的胆汁纳入研究,57.5%的胆汁标本细菌培养呈阳性。部分无感染及胆道梗阻的胆汁中仍有部分存在细菌,但细菌检出率较有感染以及胆道梗阻者明显减低,而肿瘤位置同细菌的检出率之间并无统计学关联。许多种细菌可出现在胆汁中,常见细菌依次为肠球菌属、大肠埃希菌、葡萄球菌属、肺炎克雷伯菌、铜绿假单胞菌等。而万古霉素、莫西沙星、亚胺培南等多种抗生素均对细菌具有良好的敏感性。结论:胆管癌患者无论是否存在感染及梗阻,胆汁内都有可能存在细菌,细菌的检出率同肿瘤部位无关。而肠道菌群仍是胆道细菌的主要来源。     

Specific genomic alterations and prognostic analysis of perihilar cholangiocarcinoma and distal cholangiocarcinoma

BackgroundCholangiocarcinoma (CCA), which consists of intrahepatic CCA (iCCA), perihilar CCA (pCCA), and distal CCA (dCCA), is an aggressive malignancy worldwide. PCCA and dCCA are often classified as extrahepatic CCA (exCCA). However, the differences in mutational characteristics between pCCA and dCCA remain unclear.MethodsDeep sequencing targeting of 450 cancer genes was performed for genomic alteration detection. The tumor mutational burden (TMB) was measured by an algorithm developed in-house. Correlation analysis was conducted using Fisher’s exact test.ResultsFGFR2 and ERBB2 mutations mainly occurred in iCCA and exCCA, respectively. In exCCA, the frequencies of PIK3CA, FAT4, KDM6A, MDM2, and TCF7L2 mutations were significantly higher in pCCA compared to dCCA, while the frequencies of TP53 and KRAS mutations were markedly lower in pCCA than those in dCCA. The prognosis-related mutations were different among the CCA subtypes. NF1 mutation was associated with short disease-free survival (DFS) and overall survival (OS), and ERBB2 mutation was associated with short DFS in dCCA patients. Meanwhile, MAP2K4 mutation was associated with long DFS and OS, and TERT mutation was associated with short DFS in pCCA. A series of mutations in genes, including ARID1A, ARID2, SMAD4, TERT, TP53, and KRAS, were found to be associated with the TMB.ConclusionsIn this study, we investigated the comprehensive genomic characterizations of CCA patients, identified the significant alterations in each subtype, and identified potential biomarkers for prognosis prediction. These results provide molecular evidence for the heterogeneity of CCA subtypes and evidence for further precision targeted therapy of CCA patients.     

Conformal therapy of locally advanced cholangiocarcinoma of the main bile ducts]

PURPOSE: Retrospective study of 23 patients treated with conformal radiotherapy for a locally advanced bile duct carcinoma. PATIENTS AND METHODS: Eight cases were irradiated after a radical resection (R0), because they were N+; seven after microscopically incomplete resection (R1); seven were not resected (R2). A dose of 45 of 50 Gy was delivered, followed by a boost up to 60 Gy in R1 and R2 groups. Concomitant chemotherapy was given in 15 cases. RESULTS: Late toxicity included a stenosis of the duodenum, and one of the biliary anastomosis. Two patients died from cholangitis, the mechanism of which remains unclear. Five patients are in complete remission, six had a local relapse, four developed a peritoneal carcinosis, and six distant metastases. Actuarial survival rate is 75%, 28% and 7% at 1, 3 and 5 years, respectively (median: 16.5 months). Seven patients are still alive with a 4 to 70 months follow-up. Survival is similar in the 3 small subgroups. The poor local control among R0N+ cases might be related to the absence of a boost to the "tumor bed". In R1 patients, relapses were mainly distant metastases, whereas local and peritoneal recurrences predominated in R2. CONCLUSION: Conformal radiochemotherapy delivering 60 Gy represents a valuable palliative approach in locally advanced biliary carcinoma.     

An ultrastructural morphometric analysis of normal human mammary tissue and human breast cancer

An ultrastructural morphometric study of normal and tumourous adult human mammary tissue is presented. The data show a characteristic numerical pattern for the different neoplastic stages of breast tissues. By applying a special data-comparison program a standardized diagnostic and prognostic system appears possible through the morphometric analysis of ultrathin epon sections of surgically obtained material.     

Comprehensive allelotyping of human intrahepatic cholangiocarcinoma.

We performed a genome-wide scan for loss of heterozygosity (LOH) in 22 intrahepatic cholangiocarcinoma (ICC) cases using 168 polymorphic microsatellite markers throughout all of the human chromosomes and 48 markers of which LOH is reportedly characteristic of hepatocellular carcinoma (HCC). Markers with LOH in more than 30% of informative cases were observed at 21 loci. Among these, eight markers on 6q (three loci), 4q (two loci), 9q, 16q, and 17p shared high frequencies of LOH with HCC in our previous study. As for gross appearance, mass-forming type tumors showed higher frequency of LOH (P < 0.001) compared with other types. Compared by tumor size (< or =5 cm versus >5 cm), number (multiple versus solitary), and the International Union Against Cancer TNM classification (stage IVB versus II-IVA), LOH was observed more frequently in advanced stages (P < 0.01, respectively). However, LOH frequency does not differ regardless of lymph node status (pN0 versus pN1). Frequent LOH on 1p36 including the p73 locus was noted in large tumors without lymph node metastasis. These suggest that ICC shares some common carcinogenic steps with HCC such as LOH of 4q and 6q and that inactivation of tumor suppressor genes on chromosome 1p36 contributes to progression of ICC but not to metastatic traits.     

正常人舌脱落上皮细胞的形态计量分析

获取正常人舌脱落细胞中不同细胞的形态计量学参数值及总体细胞形态计量学参数的频率分布图。采集 30例正常人舌脱落上皮细胞 ,涂片后作巴氏染色 ,应用MIPS - 1图像分析系统对脱落细胞进行形态计量学检测。测得了中层细胞、角化前细胞、不全角化细胞、完全角化细胞等不同脱落细胞的面积、周长、形状因子、等效直径和最大直径等形态计量学参数 ;分析了总体脱落细胞面积、形状因子及最大直径 3项参数。获得了正常人舌脱落细胞中不同细胞的形态计量学参数及总体细胞形态计量学参数值的频率分布图。     

内镜逆行胰胆管造影术胆道引流在胆总管恶性肿瘤及肝门部胆管癌外科围术期中的应用

目的探讨内镜逆行胰胆管造影术(ERCP)胆道引流在胆总管恶性肿瘤及肝门部胆管癌外科围术期中的应用价值。方法选取2016年10月至2018年12月间山东省立第三医院收治的行ERCP术的88例肝门胆管癌患者,根据胆道引流方式不同分为内镜下鼻胆管引流术(ENBD)组(27例)、胆道塑料支架引流术(ERBD)组(35例)与胆道金属支架引流术(EMBE)组(26例)。比较各组患者的手术情况、肝功能指标、并发症发生情况以及生存率。结果所有患者共行ERCP术93次,插管成功率为94. 6%(88/93),引流总成功率为92. 0%(81/88)。ENBD组引流成功23例,失败4例,引流成功率为85. 2%(23/27),ERBD组引流成功33例,失败2例,引流成功率为94. 3%(33/35),EMBE组引流成功25例,失败1例,引流成功率为96. 2%(25/26),三组比较,差异无统计学意义(P> 0. 05)。治疗后,三组患者的TBIL、DBIL、AST、ALT、GGT和ALP水平均较治疗前降低,差异均有统计学意义(均P <0. 05)。有3例患者术后发生胆管炎,2例患者发生消化道出血,2例患者发生胆道感染,1例患者发生急性胰腺炎,1例患者发生胆漏,并发症总发生率为10. 2%。2例行胆道支架引流患者术后出血腹痛,对症治疗后缓解。88例患者有5例患者失访,随访率为94. 3%。随访时间为1年,共有38例患者死亡,其余45例患者于随访结束时仍生存。胆总管恶性肿瘤与肝门胆管癌患者ERCP术后的3个月生存率为88. 0%,6个月生存率为68. 7%,1年生存率为54. 2%。结论ERCP胆道引流治疗胆总管恶性肿瘤和肝门部胆管癌手术成功率较高,术后并发症较少,结合不同的胆道引流术可以有效改善患者肝功能,延长生存时间。     

Microsatellite instability in thorotrast-induced human intrahepatic cholangiocarcinoma

Thorotrast, a colloidal suspension of radioactive (232)ThO(2) that emits alpha particles, was used as a radiographic contrast during World War II. It is known to induce liver cancers, most frequently ICC, decades after injection. Since radiation induces genomic instability, we analyzed MSI in Thorotrast-induced ICC. The frequency of MSI(+) cases was 62.5% in Thorotrast ICC, whereas it was 22.7% in non-Thorotrast ICC. However, frameshift mutations of mononucleotide repeats were not observed in Thorotrast ICC. In addition, the MSI(+) phenotype was not associated with the quantity of Thorotrast deposited or the latency period of ICC induction. Promoter regions of both the hMLH1 and the hMSH2 MMR genes tended to be hypermethylated in the tumor part compared to the adjacent nontumor part in Thorotrast ICC. Methylation of the hMLH1 promoter was associated with the MSI(+) phenotype in Thorotrast ICC. In contrast, methylation status of these promoter regions was not related to MSI in non-Thorotrast ICC cases. These findings suggest that MSI induced by exposure to Thorotrast mainly reflects clonal expansion of cancer cells and is partly due to inactivation of hMLH1 by hypermethylation.