肝细胞癌中差异表达N-连接糖蛋白的分析鉴定

目的分析鉴定与肝细胞癌(HCC)发生发展相关的差异表达的N-连接糖蛋白。方法应用刀豆蛋白凝集素(ConA)、晶状体凝集素(LCH)和雪花凝集素(GNA)组成的亲合层析柱富集10对HCC和癌旁非癌组织中N-连接糖蛋白、二维电泳(2DE)比较分析差异表达的蛋白质点、串联质谱鉴定差异表达蛋白,Western blotting验证人羧酸酯酶1(hCE1)、触珠蛋白(HP)及组织蛋白酶D(CD)的差异表达;体外侵袭实验检测CD表达沉默后对HCC的侵袭力影响。结果质谱、生物信息学等技术鉴定了28个与HCC相关的差异表达蛋白(HCC中高表达和低表达的蛋白均为14个),Western blotting验证hCE1、HP在HCC中明显低表达,组织蛋白酶D前体(pCD)在HCC中高表达;而ConA亲和层析柱富集的ConA-CD在HCC中显著高表达。CD-siRNA介导的CD表达沉默能够显著降低肝癌细胞系SNU449、SNU473的体外侵袭能力。结论 HP、hCE1蛋白表达变化和CD N-糖链变异可能参与了HCC发生发展过程。     

P-糖蛋白在肝细胞癌中的表达及临床研究

目的：探讨Ｐ－糖蛋白在细胞癌组织中表达及临床意义。方法：采用免疫组化法对７２例肝细胞癌，９例正常肝组织进行Ｐ－糖蛋白测定，作相关临床因素分析。结果：肝细胞癌组织中Ｐ－糖蛋白的阳性表达率为４４．０％，正常肝组织表达率为２２．０％（Ｐ〈０．０５）。Ｐ－糖蛋白的表达与肝癌的组织分型、分级及预后无明显相关性，并且化疗并不影响Ｐ－糖蛋白阳性表达病人的预后。结论：Ｐ－糖蛋白在肝细胞癌中的过表达，提示其在肝细胞     

肝细胞癌患者血清蛋白质组成分的双向凝胶电泳-飞行时间质谱分析

目的　分析肝细胞癌患者与正常人血清双向凝胶电泳的差异蛋白质 ,寻找肝癌早期血清学诊断的可能指标。方法　固相 pH梯度双向凝胶电泳分离肝细胞癌患者及正常人血清总蛋白。银染显色 ,PDQuest 2DE软件分析 ,对差异蛋白质点用基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)测定其胶内酶解后的肽质指纹图谱 ,用PepIdent软件查询SWISS PROT数据库。 结果　获得了重复性较好的双向电泳银染图谱。图像分析检测 3块胶平均匹配的点数占平均蛋白点数的匹配率是 70 2 %。等电聚焦方向的平均偏差为 (1 0 2± 0 2 2 )mm ,十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)方向的平均偏差为 (0 97± 0 14 )mm。将 2 3个差异点进行胶内原位酶解 质谱指纹图分析 ,获得 15张肽质指纹图 ,查询数据库进行了初步鉴定。结论　固相 pH梯度双向凝胶电泳分离人血清总蛋白获得重复性较好的结果 ,但仍需进一步探索如何去除高丰度已知蛋白及脂类等物质的方法 ,以便得到分辨率更高的双向电泳银染图谱。MALDI TOF MS肽质指纹图分析鉴定的结果为人肝细胞癌血清学诊断指标的筛选提供了依据     

乙肝病毒相关原发性肝细胞癌的癌前标志物检测

探讨乙肝病毒（HBV）相关原发性肝细胞癌（HCC）癌前标志物的检测及其临床意义。在HBx基因转染的HepG2细胞系中,采用PCR选择性分离cDNA,进行差减杂交,得到5个HBxAg上调基因URG4、URG7、URG11、S15a、Sui1。建立特异ELISA方法,对中国居民及美国的韩国裔移民共1046例（其中慢性HBV感染400例,对照组646例）,检测5个HBxAg上调基因的相应抗体,定其为HBV相关HCC癌前标志物,简称癌前抗体。结果表明,慢性乙肝组、乙肝肝硬化组、HBV相关HCC组以及HCV相关HCC组检出癌前抗体的阳性率与正常人群相比均有显著差异（P〈0．01）,且差异随慢性乙肝-乙肝肝硬化-肝癌的发展而增大,而HBV携带组、其它肝炎组以及其他肿瘤组与正常人群相比均无显著差异（P〉0．05）。在5个癌前抗体中,HBV相关HCC组检出3个或3个以上癌前抗体的阳性率为52．6％（30／57）,乙肝肝硬化组为29．2％（35／120）,慢性乙肝组为19．0％（29／153）,而正常人群仅为0．8％（4／494）。在HBV感染各组病例中,慢性乙肝组癌前抗体阳性率为48．4％（74／153）,乙肝肝硬化组为66．7％（80／120）,HBV相关HCC组为82．5％（47／57）（P〈0．01）,平均癌前抗体阳性数分别为1．37、2．12和3．52（P〈0．01）。这5个癌前抗体在正常人群中检测不到,而在肝癌及其高危人群中高比例地出现,且阳性率随慢性乙肝-乙肝肝硬化-肝癌的发展而显著升高,反映了这些癌前抗体是肝癌发生的高危风险因子,在肿瘤的发展过程中逐步产生,可以作为癌前病变趋势的诊断依据。     

肝细胞癌中c-myc和c-fos表达及其意义

目的探讨c-myc和c-fos在肝细胞癌(HCC)中的表达及其意义。方法采用免疫组化方法检测分析10例正常肝组织及81例HCC癌旁肝组织和癌组织中c-myc和c-fos蛋白的表达。结果 (1)正常肝组织中c-fos和c-myc均无阳性表达;而在癌组织与癌旁组织则出现阳性表达,其中癌组织的阳性表达率分别为41.9%(34/81)和39.5%(32/81),癌旁组织则为14.8%(12/81)和18.5%(15/81),差异具有统计学意义(P0.05)。(2)c-myc和c-fos在癌组织中的表达与肿瘤分级及分化程度相关(P0.05),但与年龄、性别、肿瘤直径、是否伴有肝硬化及HBsAg阳性无关(P0.05)。结论 c-myc和c-fos的阳性表达与HCC的发生相关。     

肝细胞癌抗原587基因甲基化调节HCA587-TAF9互作介导肝细胞癌的侵袭和迁移

目的：研究肝细胞癌抗原587（HCA587）在肝细胞癌（HCC）组织中的表达和启动子区甲基化状态,探 讨HCA587对肝细胞癌细胞迁移和侵袭的调节机制。方法：纳入肝细胞癌肿瘤组织114 例,分为HCC组和癌旁 非癌组织（NT）组。荧光定量PCR 检测HCA587的mRNA表达量;免疫组织化学和免疫印迹检测HCC组和NT 组组织中HCA587的蛋白表达。基于甲基化敏感酶和甲基化依赖酶酶切,并结合荧光定量PCR 方法分析肝细胞 癌组织中HCA587基因启动子区甲基化状态。构建重组HCA587过表达质粒（pcDNA3.1-HCA587）,培养人肝 细胞癌细胞系BEL-7404,转染pcDNA3.1-HCA587 或者使用甲基化抑制剂5-Azacytidin 处理细胞。细胞分为对照 组、5-Azacytidin 组、pcDNA3.1-HCA587 组和pcDNA3.1-HCA587 空载体（EV） 组。免疫沉淀法分析HCA587 与TATA 盒结合蛋白相关因子9（TAF9）的结合,Transwell 小室检测细胞的迁移和侵袭能力。结果： 与癌旁非 癌组织组比,HCC组的HCA587在mRNA和蛋白水平均上调。免疫组织化学证实HCC组组织中HCA587阳性表 达。HCC组中HCA587的启动子区CpG 岛甲基化水平降低。体外实验结果显示,5-Azacytidin 促进BEL-7404 中 HCA587的表达、HCA587与TAF9的结合、BEL-7404 细胞的迁移和侵袭能力,但抑制HCA587甲基化水平;另外, HCA587过表达增强BEL-7404 细胞的迁移和侵袭能力。结论：HCA587高表达或抑制其启动子区甲基化可以促进 HCA587与TAF9的结合及导致肝细胞癌细胞的迁移和侵袭。     

肝细胞癌相关基因的研究进展

肝细胞癌（HCC）是死亡率仅次于胃癌、食道癌的第3大常见恶性肿瘤,其发病率呈逐年上升趋势.HCC属于多因素复杂疾病,是病毒因素、环境因素和机体的遗传因素交互作用,经过多个阶段的发展而产生的.尽管近年来HCC的治疗有了很大的进展,但在临床上肿瘤的复发和转移频繁发生,HCC患者的5年生存率依然很低[1].肿瘤相关基因（癌基因或抑癌基因）是在肿瘤中突变或表达异常的一类分子,它们的改变会引起细胞过度增殖、凋亡抑制等生物学效应,从而导致肿瘤的发生.研究特定基因及其表达产物与HCC发病的关系,对于更好的了解HCC发病机制及进行诊疗,有着重要的意义.     

肝细胞癌中ZHX2、NF-YA及AFP表达的相关关系

目的 检测肝细胞癌(hepatocellular carcinoma,HCC)组织中ZHX2、NF-YA及AFP mRNA和蛋白表达的相关关系.方法 采用RT-PCR及免疫组织化学方法检测HCC组织中ZHX2、NF-YA及AFP mRNA和蛋白的表达情况.结果 ZHX2 mRNA在AFP mRNA阴性的25例HCC组织中的表达率为52.0%,明显高于AFP mRNA阳性组织中的表达率(26.3%,P=0.038),ZHX2 mRNA与AFP mRNA表达呈负相关(OR=0.33);NF-YA mRNA与ZHX2 mRNA表达呈正相关(OR=31.429),而与AFP mRNA表达呈负相关关系(OR=0.308).AFP蛋白阴性的142例HCC组织中,ZHX2蛋白的表达率为23.9%,明显高于在AFP蛋白阳性组织中的表达率(12.8%,P=0.034);HCC组织中ZHX2蛋白与AFP蛋白表达呈负相关(P=0.018,r=-0.153).NF-YA蛋白在HCC组织中与ZHX2蛋白表达呈正相关(P=0.000,r=0.371),与AFP蛋白表达呈负相关(P=0.000,r=-0.497).结论 联合检测AFP和ZHX2蛋白将提高HCC的诊断率;ZHX2可能是通过NF-YA来调控AFP的表达.     

双向电泳在疾病研究中的应用

生命的表现形式最终是由蛋白质决定的。因此,对蛋白质组的研究具有很现实的意义。双向电泳(2-DE)技术作为蛋白质组学研究的一项核心技术,主要用于细胞、组织以及其他样本蛋白质提取物的分析。近年来,2-DE结合质谱(MS)技术被广泛用于差异蛋白的鉴定、疾病标志物的筛选、药物靶标的确定等,目前已经成为蛋白质组学研究的支撑技术,以其高通量、高分辨率和重复性被广泛应用到各个领域,特别是生物医学的研究中。本文就近年2-DE技术的应用进展,尤其在生物医学方面的贡献做一综述。     

Carcinoembryonic antigen and lectin binding in the bile canalicular structures of hepatocellular carcinoma

Summary Bile canalicular structures of 32 hepatocellular carcinomas and 36 cirrhotic livers were investigated by light microscopy using immunohistochemistry for carcinoembryonic antigen (CEA) and lectin histochemistry. CEA positive bile canalicular structures were found in 17 out of 32(53%) hepatocellular carcinomas and in 33 of 36(92%) cirrhotic livers. Among the 10 lectins examined, about 40% of the CEA positive bile canalicular structures of hepatocellular carcinomas showed positive binding of MPA, DBA, WGA, RCA-I or UEA-I, whereas only MPA or RCA-I bound to the CEA positive bile canalicular structures of cirrhotic liver, in about 20% of cases. It has been shown that the CEA positive bile canalicular structures of hepatocellular carcinomas are heterogeneous and differ from those of cirrhotic liver in lectin histochemistry.Presented at the XVI International Congreess of International Academy of Pathology (Rikuo Machinami 1986)     

Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry

Despite improvements in treatment of patients with head and neck squamous cell carcinoma (HNSCC) over the last two decades, the survival rate of these patients has not increased significantly. One of the major factors in the poor outcome of the disease is regional metastasis. To better understand the mechanisms of this process at the protein level, we performed two-dimensional electrophoresis (2-DE) and mass spectrometry using SELDI ProteinChip technology to identify proteins differentially expressed in two HNSCC cell lines, UMSCC10A and UMSCC10B, from the same patient. UMSCC10A was derived from the primary tumor and UMSCC10B from a metastatic lymph node. The differentially expressed proteins were excised from the gels. Following in-gel digestion by trypsin, mass profiles of the peptides were generated. Proteins were identified by submitting the peptide mass profiles to a public available NCBInr databases (www.proteometrics.com). Two membrane-associated proteins, annexin I and annexin II, and glycolytic protein enolase-α were found to be upregulated, and calumenin precursor down-regulated, in metastatic cell line UMSCC10B. The identity of these proteins was confirmed by analyzing additional peptide mass fingerprints obtained by endoproteinase lysine-C digestion. The results were also validated by Western blotting analysis. Our results showed that enolase-α, annexin-I and annexin-II might be important molecules in head and neck cancer invasion and metastasis. The results also suggest an important complementary role for proteomics in identification of molecular abnormalities important in cancer development and progression. This revised version was published online in July 2006 with corrections to the Cover Date.     

肝癌抗原特异性的检测

目的　检测两种新的人肝细胞癌 (HCC)抗原 (编号为HCC 1 8a和HCC 3 13)对HCC的特异性。方法　应用重组克隆表达抗原的血清学技术 (SEREX) ,用异体HCC血清和非HCC血清检测两种新的HCC抗原的特异性。结果　相应的抗体主要见于HCC患者 ,阳性反应率分别为 80 % (8/ 10 )及 90 % (9/ 10 ) ;胃癌为 0及 2 0 % (1/ 5 ) ;正常人各为 7.1% (2 / 2 8) ;乳癌为 0。结论　两种新的HCC抗原对HCC有高度特异性 ,可考虑作为HCC血清学诊断的指标之一。     

腹主动脉狭窄致大鼠心肌肥大后心肌组织蛋白质组变化的研究

目的探讨腹主动脉狭窄诱导大鼠心肌肥大后心肌组织蛋白质表达谱的变化。方法健康雄性Wistar大鼠12只,随机分为模型组和对照组（均n=6）。模型组行腹主动脉缩窄术,对照组仅分离腹主动脉不行缩窄术。术后4周结束实验并提取左心室组织蛋白质,双向电泳分离心肌组织蛋白质,分析差异显示的蛋白质并选取9个差异显著的蛋白点进行胶内酶切和质谱分析。结果双向电泳可分离（454±13）个蛋白质,点匹配率为（78．7±1．4）％,心肌组织13种蛋白质出现明显差异表达,与对照组比较,模型组4种蛋白质表达上调,9种蛋白质表达下调。质谱分析鉴定出7个蛋白质为：与能量代谢相关的丙酮酸脱氢酶E1和β-烯醇酶、与细胞收缩功能相关的α-肌球蛋白重链和心脏α-肌动蛋白前体、与甲状腺激素代谢相关的甲状腺素结合蛋白等5种蛋白质在模型组表达下调,肌球蛋白轻链和与细胞内源性保护相关的热休克蛋白B6等2种蛋白质在模型组表达上调。结论腹主动脉缩窄致大鼠心肌肥大后,心肌组织蛋白质组变化显著,这些变化涉及与心肌细胞能量代谢、细胞骨架以及心肌收缩等有关的几组蛋白质,提示上述蛋白质组改变可能参与了心肌肥大过程。     

Identification of Epicoccum purpurascens allergens by two-dimensional immunoblotting and mass spectrometry

Sensitization to allergens of Epicoccum purpurascens is reported from many places world over. Previously, the IgE binding proteins of Epicoccum were studied by one-dimensional immunoblotting (1-DE). However, the allergens recognized by 1-DE may be a mixture of proteins appearing at the same molecular weight. In the present study, IgE-binding components of E. purpurascens were detected using two-dimensional immunoblotting and mass spectrometry. Approximately 250 protein spots were identified by Coomassie staining in the range of 14-110kDa and pI 3.5-9.5. Of these, 147 showed specific IgE reactivity with pooled patients' sera whereas 16 protein components reacted with more than 50% of the individual patients' sera. The frequency of IgE reactivity was highest (87.5%) for a 25-kDa protein (pI-7.85). Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of 16 protein components led to identification of 6 important allergens as phospho-glycerate kinase (PGK), RNA-dependent RNA polymerase, pyrroline-5-carboxylate dehydrogenase, 40S ribosomal protein and two proteins of unknown functions. The major allergens identified may be assessed for cross reactivity with other fungi. In conclusion, 2-DE followed by immunoblotting allowed characterization of E purpurascens allergens. Six new allergens have been identified using this technique and mass spectrometry. The availability of such relevant allergens would help in component-based diagnosis and therapy of fungal allergy.     

Towards a global analysis of porcine alveolar macrophages proteins through two-dimensional electrophoresis and mass spectrometry

Alveolar macrophages (AM) are the primary phagocytes of the innate immune systems, constituting a link between innate and adaptive immunity. With the aim of studying the porcine AM biology and the dynamics of pig-pathogen cell interactions, we have obtained a reference 2-DE map of the porcine AM proteins. The proteins were separated by 2-DE using a 5–8 range pH gradient in isoelectric focusing and over 800 spots were detected. A set of proteins, covering the pI 5.2–7.4 and M_W 19 to 106 kDa ranges, was subjected to MS analysis and 106 proteins were assigned identification by PMF, this identification being confirmed by MS/MS. An important number of proteins is involved in immunological functions, signalling process, transport or apoptosis, confirming that macrophages are involved in a wide range of biological functions. This reference map provides a useful tool for identifying protein pattern changes as a result of inflammation, exposure to infectious agents or genetic diseases.     

Identification of two naturally presented MAGE antigenic peptides from a patient with hepatocellular carcinoma by mass spectrometry

In the absence of efficient systemic chemotherapy, immunotherapy is considered a hopeful treatment for controlling recurrence of hepatocellular carcinoma (HCC). The identification of proper antigenic peptides presented by MHC class I molecules is a critical step for the development of therapeutic vaccines against tumors. Currently, the "reverse immunology" approach is the most commonly used technique in the identification of the tumor-associated T cell epitopes. However, it is based on T cell dependent approach and cannot fully reflect the actual presentation of epitope in tumor in vivo. In the present study, we managed to identify the naturally presented MAGE epitopes of HCC directly by epitope prediction, HPLC differential analysis and MS detection. We successfully detected a naturally processed peptide FLWGPRALV (MAGE-3(271-279), HLA-A2-restricted) with an estimated number of 38-39 copies/cell in HCC. To our knowledge, this is the first evidence that the naturally processed MAGE-3(271-279) can be isolated and identified from the tumor tissue of HCC patient. Furthermore, specific CD8(+) T cell responses to this epitope were also found after tumor relapse by IFN-gamma release Cytospot and tetramer assay indicating that MAGE-3(271-279) was indeed presented by HCC cells in vivo. In addition, another new antigen peptide was found, which may be derived from MAGE-1. Our findings demonstrate the potential of the direct approach for identification of tumor-associated epitopes. This approach may become a useful tool for the development of vaccine against cancer in the future.