消化系统肿瘤转移相关蛋白质组学的研究进展

蛋白质组学已经成为当今研究肿瘤转移领域的核心技术之一，在筛选肿瘤转移相关蛋白方面中取得了许多进展。通过蛋白组学技术可以找到肿瘤转移相关蛋白，不仅有助于揭示肿瘤发生、发展的分子机制，为肿瘤转移诊断和预后预测提供生物学标志物，还可以为肿瘤治疗提供靶点。     

卵巢癌的蛋白质组学研究进展

目前蛋白质组学已在卵巢癌的多方面研究中得到广泛的应用，如对卵巢癌发病机制的研究、卵巢癌生物标记物的筛选、卵巢癌信号转导通路的研究及协助诊断、协助卵巢癌分类、寻找治疗靶点等。随着人们对蛋白质组学认识的深入,卵巢癌发生发展的机制将会日渐明了。     

蛋白质组学方法鉴定小鼠Barnes迷宫空间记忆相关蛋白

目的：通过Barnes Maze （ Barnes迷宫, BM）对小鼠进行空间记忆训练,随后用蛋白质组学方法鉴定BM空间记忆相关的海马蛋白并分析蛋白功能及参与的生物学过程。方法将小鼠分为实验组和对照组。应用BM对实验组小鼠进行空间记忆训练,蛋白质组学方法鉴定两组小鼠中表达水平不同的海马蛋白,分析空间记忆相关的蛋白及其功能。结果实验组小鼠学习训练后学习记忆能力明显强于对照组,到达目标孔的时间明显缩短[对照组：（116．0±5．5） s,实验组：（42．0±3．6） s; P＜0．05]。蛋白质组学分析结果表明9种海马蛋白的表达水平在记忆形成过程中显著下调,下调的蛋白按功能可分为4类：①细胞骨架;②能量代谢;③神经发育;④物质运输。结论这9种学习记忆相关蛋白可能通过调节细胞骨架、能量代谢、神经发育和物质运输等生物学过程进而影响了学习记忆能力。     

胰腺癌转移相关基因C14orf166的真核表达及其蛋白相互作用的蛋白质组学筛选

目的旨在对胰腺癌转移相关基因C14orf166进行真核重组表达,并在此基础上结合蛋白质组学方法初步筛选其相互作用蛋白,为进一步研究C14orf166在肿瘤转移中的作用提供研究基础。方法构建人C14orf166的带双标签的真核表达载体pcDNA3.1-Flag-C14orf166-His,通过脂质体将其转染至人胚肾293T细胞。采用Ni-agrose进行His标签蛋白的pull-down纯化,分离C14orf166的蛋白结合复合体,对蛋白混合物进行SDS-PAGE分析,选择差异条带,进行MALDI-TOF-TOF质谱鉴定。结果在293T细胞中重组表达了C14orf166蛋白,对C14orf166蛋白复合体进行分离鉴定后,筛选出RS8、EFCB9和NRAP3个可能与C14orf166相互作用的蛋白。结论基因重组表达结合pull-down和蛋白质组学方法可以发现新的相互作用蛋白,为进一步了解C14orf166在肿瘤发病机制中的作用提供了新的线索。     

木犀草素对卵巢癌细胞株转移能力的影响

目的：研究木犀草素对卵巢癌细胞株HO-891 0PM的体外侵袭能力的影响，并探讨其可能的作用机制。方法：HO-8910P M细胞经木犀草素处理后用Boyden小室法检测其体外、侵袭和运动能力。并采用明胶酶 谱法检测HO-8910PM细胞分泌的明胶酶活性。RT-PCR检测TIMP-1、NM23基因mRNA的表达情况，蛋白印迹法检测ERK2的蛋白表达变化。结果：HO-8910PM细胞经木犀草 素处理后，体外侵袭、运动能力呈剂量依赖性下降。HO-8910PM细胞MMP-9的分泌下降。ERK2 蛋白表达明显降低，但TIMP-1、NM23基因mRNA的水平无明显变化。结论:木犀草素在体外剂量依赖性地抑制卵巢癌细胞HO-8910PM的转移能力，这可能与木犀草素 抑制MMP-9的分泌及下调ERK2表达有关。     

卵巢癌转移灶细胞粘附分子检测及其腹腔播散机制探讨

目的;探讨上皮钙依赖粘附素（Ｅ－ＣＤ）及ＣＤ４４等细胞粘附分子在卵巢癌腹腔播散中的作用。     

磷脂酶A_2抑制剂对卵巢癌细胞转移潜能的影响

目的:卵巢癌细胞可分泌大量溶血性磷脂酸(LPA),LPA促进卵巢癌的发展,磷脂酶A2是LPA合成的主要酶。拟研究磷脂酶A2的抑制剂能否抑制卵巢癌的转移。方法:Transwell检测细胞迁移能力变化;Western blot检测磷脂酶A2的表达/活化状态;ECIS检测细胞浸润能力;质谱分析LPA浓度;裸鼠卵巢癌模型检测肿瘤的转移。结果:在体外LPA以剂量依赖方式促进SKOV3细胞的迁移和黏附;SK-OV3细胞内具有高表达和活化的iPLA2和cPLA2酶;应用AACOCF3和HELSS后,SKOV3分泌的LPA显著减少;AA-COCF3和HELSS显著抑制裸鼠内SKOV3的远地转移。结论:LPA显著促进SKOV3的迁移、黏附、对单层腹膜的浸润。AACOCF3和HELSS显著抑制SKOV3分泌LPA、并显著抑制SKOV3的迁移和浸润,还抑制裸鼠内SKOV3的远地转移,AACOCF3和HELSS有可能成为卵巢癌转移的潜在抑制剂。     

高转移人卵巢癌裸鼠皮下移植瘤模型的建立及其生物学特性

用人卵巢癌细胞系HO－8910移植探鼠，建立高转移人卵巢癌探鼠皮下移植瘤模型命名为（NSMO），已传代23次，仍保持高转移的特性。共接种BALB/c棵小鼠57只，皮下成瘤率为100％，平均带瘤存活时间为159．9天。在解剖47只裸鼠中有转移瘤鼠42只（89．4％）。最早出现转移的时间为56天。移植瘤组织学和超微结构保持原来人卵巢分化差的浆液性乳头状囊腺癌的形态特征和分泌功能。流式细胞术分析显示DNA指数为1．4，染色体分析显示众数为54（属于超二倍体），显示人类癌症的特征。移植瘤相关标记物检测显示大多数癌细胞雌激素受体和孕激素受体阳性。该模型为转移机制的研究和寻找抗转移药物提供了理想的工具。     

蛋白质组学在肿瘤研究中的应用

肿瘤是机体正常细胞在多因素、多基因作用下,经过多个步骤发生转化,失去原有的生长分化调节而异常增生的结果。因此,肿瘤细胞与其起源的正常细胞相比,蛋白质既有相同也有不同点。应用蛋白质组学研究技术可以比较患有同一肿瘤的不同个体的肿瘤组织(或细胞)之间,或者同一个体肿瘤细胞与正常起源细胞之间蛋白质在表达数量、表达位置和修饰状态上的差异,可以发现肿瘤发生过程中蛋白质变化的规律,筛选和确定肿瘤特异性标记物,对肿瘤发病机制的探讨以及在其诊断和有效治疗均具有重要的意义。     

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.     

Claudin proteins in ovarian cancer

Members of the claudin family of tight junction proteins have been found altered in several malignancies, including ovarian cancer. Because claudin-3 and -4 are elevated in the vast majority of ovarian tumors, they may represent useful biomarkers for detection and prognosis, as well as ideal targets for therapy using the Clostridium perfringens enterotoxin.     

蛋白质组学检测结直肠腺瘤及早期恶变患者血清的差异蛋白质变化及意义

目的： 通过比较分析结直肠腺瘤及早期恶变患者血清蛋白质的表达差异,寻找结直肠癌早期诊断的血清标记物。方法：建立结直肠腺瘤及腺瘤早期恶变患者血清蛋白双向电泳图谱,比较分析差异蛋白质斑点,切取酶解行MALDI-TOF／TOF质谱分析鉴定。结果：成功建立结直肠腺瘤及早期恶变患者血清蛋白双向电泳图谱,其平均蛋白质点分别为1672±73和1732±46,早期恶变组表达差异大于1.5倍的蛋白质斑点有28个,其中15个下调,13个上升;质谱分析鉴定出23个蛋白质,鉴定率达82.14%;合并重复鉴定的蛋白质,共获得的差异蛋白13种,其中8种为上调蛋白,5种为下调蛋白,分为6类,包括蛋白酶抑制剂、补体系统、免疫球蛋白、角质素、信号转导蛋白及未知蛋白。结论：蛋白质组学技术能灵敏分析结直肠腺瘤及早期恶变患者血清蛋白质的异常改变,本研究鉴定的蛋白质可能成为结直肠癌早期诊断的血清肿瘤标记物。     

Identification of proteins regulated by estradiol in focal cerebral ischemic injury—A proteomics approach

Estradiol protects neuronal cells against permanent and focal ischemic brain damage. We identified the proteins that are expressed following estradiol administration during cerebral ischemia in an animal model. Adult female rats were ovariectomized and treated with oil or estradiol prior to middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, and brains were collected 24 h after MCAO. Protein analysis was performed on the cerebral cortex using two-dimensional gel electrophoresis. Protein spots with difference in intensity between oil- and estradiol-treated groups were identified by mass spectrometry. Among these proteins, levels of protein phosphatase 2A (PP2A) and astrocytic phosphoprotein PEA-15 were significantly decreased in the oil-treated group in comparison to the estradiol-treated group. Moreover, Western blot analysis demonstrated that estradiol treatment prevents injury-induced decrease of PP2A and PEA-15 levels during both MCAO-induced injury and glutamate exposure in HT22 cells. In contrast, levels of the 60 kDa heat shock protein (Hsp 60) were significantly increased in oil-treated animals, while estradiol prevented the injury-induced increase of Hsp 60. The results of this study provide an evidence that estradiol protects neuronal cells against ischemic brain injury through the up- and down-modulation of specific proteins.     

用基因芯片研究人肺癌转移相关基因

目的 自制肿瘤转移相关基因芯片研究人肺癌高、低转移细胞系PG和PAa间以及肺癌、淋巴结转移癌与正常肺组织间的基因表达谱差异，筛选与肺癌转移相关的特异基因。方法 提取2种肺癌细胞系、人肺癌、淋巴结转移癌及周围正常肺组织的mRNA后逆转录标记cDNA探针，与自制的含399个肿瘤转移相关基因的微阵列膜杂交，QuantArray软件分析杂交信号强度获得差异表达基因。结果 正常肺组织与肺原发癌的基因表达谱有显著差异。淋巴结转移癌组织与PG高转移细胞系表达基因行聚类分析，共同表达且最具统计学意义的基因有64个，包括上调基因27个，下调基因37个。结论 多基因参与肺癌的转移过程，肺转移癌与高转移细胞系共同差异表达的基因可能与肺癌的高转移特性密切相关。     

Identification of metastasis-associated proteins through protein analysis of metastatic MDA-MB-435 and metastasis-suppressed BRMS1 transfected-MDA-MB-435 cells

BRMS1 (breast cancer metastasis suppressor 1) was recently identified as a novel breast cancer metastasis suppressor gene. To further characterize BRMS1-mediated metastasis suppression, we applied two-dimensional proteomic and mass spectrometry (LC-tandem MS and MALDI-TOF) analysis to identify proteins differentially expressed between highly metastatic MDA-MB-435 cells and metastasis-suppressed BRMS1-transfected MDA-MB-435 cells. Quadruplicate independent 2D gels were run and analyzed under identical conditions. Following in-gel trypsin digestion of seven differentially expressed proteins, amino acid sequence and mass profiles of the peptides were generated. Proteins were identified from the NCBI non-redundant database using the search program TurboSequest. Differential expression was confirmed for five proteins, including annexin I and alpha B-crystallin, by Northern blot analysis and immunostaining. Furthermore, we showed that both proteins were expressed in vivo in lungs containing metastasized MDA-MB-435 cells but not expressed in normal lung tissue of athymic mice. Our results suggest that annexin I and alpha B-crystallin are important cellular proteins that are down regulated through BRMS1 mediated metastasis suppression.     

人上皮性卵巢癌细胞系侧群细胞的肿瘤干细胞样细胞生物学特性及膜蛋白差异表达的鉴定

目的 鉴定上皮性卵巢癌细胞侧群细胞(SP细胞)肿瘤干细胞样细胞生物学特性,并检测侧群细胞内差异蛋白质的表达.方法 流式分选上皮性卵巢癌细胞系SKOV3和A2780具有外泌Hochest33342染料生物学特性的侧群细胞,鉴定其肿瘤干细胞样细胞相关生物学特性.运用细胞培养稳定同位素标记(SILAC)的定量蛋白质组学技术,...     

Identification of sesame seed allergens by 2-dimensional proteomics and Edman sequencing: seed storage proteins as common food allergens

BACKGROUND: Sesame seed allergy is becoming increasingly prevalent, probably because of its use in international fast-food and bakery products. Despite this fact, few studies have focused on the identification of its major allergenic proteins. OBJECTIVE: The aim of this study was to identify allergenic proteins of sesame seeds (Sesamum indicum). METHODS: Extracted sesame seed proteins were separated by means of SDS-PAGE and 2-dimensional (2-D) PAGE. Immunolabeling was performed with individual patient sera from 20 patients with sesame seed allergy. Selected proteins were further analyzed by means of Edman sequencing. RESULTS: IgE-binding proteins were identified at 78, 52, 45, 34, 32, 29, 25, 20, 9, and 7 kd. Analyzing internal sequences, the protein at 45 kd, which was recognized by 75% of the patients, was found to be a 7S vicilin-type globulin, a seed storage protein of sesame and named Ses i 3. The protein at 7 kd was found to be a 2S albumin, another seed storage protein of sesame and named Ses i 2. Seed storage proteins are known food allergens in peanut, walnut, Brazil nut, and soybean. Interestingly, one known IgE-binding epitope of the peanut allergen Ara h 1 has 80% homology with the corresponding area of Ses i 3. The different amino acids were previously shown not to be critical for IgE binding in Ara h 1. In addition, the proteins at 78 and 34 kd were found to be homologous to the embryonic abundant protein and the seed maturation protein of soybeans, respectively. CONCLUSION: The identification of 4 sesame seed allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE binding epitopes in common food allergens might be a useful tool for predicting cross-reactivity to certain foods.     

蛋白质组学技术分离及鉴定人脑氧化蛋白质

背景：氧化修饰蛋白质在脑老化及阿尔茨海默病等老年变性神经病的发病机制中扮演着重要角色。蛋白质组学技术可以发现氧化修饰蛋白质,为相关疾病的药物治疗选择新的靶标。 目的：建立以双向电泳结合Western blot及生物质谱技术分离、鉴定人脑氧化修饰蛋白质的方法。 方法：取3个无神经系统疾患的男性脑组织蛋白,第一向等电聚焦电泳后,固相pH梯度胶条同二硝基苯肼反应,然后行第二向SDS-PAGE电泳。2张相同凝胶中的1张行考马斯亮蓝染色,另1张凝胶上免疫荧光显色。同PVDF膜上显色点相对应的考染凝胶上的蛋白质点即为氧化修饰蛋白质。凝胶蛋白胶内酶解,基质辅助激光解析飞行时间串联质谱鉴定氧化修饰蛋白质。 结果与结论：从提取的脑组织中鉴定出β-肌动蛋白、天冬氨酸氨基转移酶、果糖二磷酸醛缩酶、磷酸甘油酸激酶、1,3-磷酸甘油醛脱氢酶、碳酰还原酶1、磷酸丙糖异构酶、转酮醇酶、丙酮酸激酶、血清白蛋白前体、二氢嘧啶酶相关蛋白质2、热休克蛋白60为氧化修饰蛋白质。说明实验成功建立了用蛋白质组学技术分离及鉴定人脑氧化蛋白质的方法。