前列腺癌PC-3多西紫杉醇耐药细胞株的蛋白组学分析

  目的  比较前列腺癌PC-3细胞株对多西紫杉醇(docetaxel)耐药前后的蛋白质差异性表达, 了解前列腺癌PC-3细胞株耐药性产生机制。  方法  利用逐渐加量的方式培养前列腺癌PC-3多西紫杉醇耐药细胞株, 利用双向荧光差异凝胶电泳(DIGE)定量筛选PC-3细胞敏感株与耐药株的差异蛋白, 并用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF/TOF-MS)对差异位点蛋白进行成分鉴定。  结果  利用DIGE结合MALDI-TOF/TOF-MS质谱技术分析, PC-3细胞耐药株较敏感株成功分离出49种差异表达蛋白质, 29种表达上调, 20种表达下调。其中ATP synthase、Galectin-1等参与肿瘤血管的生成, Calreticulin、CathepsinD、Coflin-1蛋白参与肿瘤的转移; 78 kDa glucose-regulated protein(GRP78)、Microtubule-associated protein-6等参与肿瘤的耐药性调节。  结论  人前列腺癌PC-3细胞株多西紫杉醇耐药前后存在蛋白质的差异性表达, 为进一步发现前列腺癌转移及耐药性的分子机制以及晚期激素非依赖性前列腺癌的靶向药物治疗提供实验依据。      

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。     

维甲酸耐药细胞与敏感细胞的差异表达蛋白分析

背景与目的:维甲酸耐药机制有待进一步的深入研究。蛋白质组学以所有蛋白质为研究对象,从细胞水平及整体水平研究蛋白质的组成及其变化规律。本实验应用蛋白组学技术整体比较维甲酸耐药细胞与敏感细胞蛋白表达谱,筛选差异表达蛋白,以期获得维甲酸耐药相关蛋白。方法:提取维甲酸耐药细胞株MR2及维甲酸敏感细胞株NB4的总蛋白,通过双向凝胶电泳分离,获得二者高质量的蛋白表达谱,经PDQuestv7.1软件比较分析,筛选出二者间差异表达的蛋白质点,通过质谱仪鉴定表达差异的蛋白。结果:获得了分辨率高、重复性好的APL细胞的双向电泳图谱。PDQuestv7.1软件分析结果表明,MR2细胞及NB4细胞的pH4～7双向凝胶电泳所展示的蛋白点分别有(890±45)个和(912±56)个。二者间有57个差异显著的蛋白点,其中23个在MR2细胞中上调,34个下调。经质谱鉴定了10个蛋白,鉴定成功率达70%,鉴定的蛋白涉及癌蛋白、细胞周期调控和信号转导相关蛋白质。结论:应用双向凝胶电泳技术整体展示了维甲酸耐药细胞株MR2及敏感细胞株NB4的蛋白表达谱,并分析、鉴定、筛选出与维甲酸耐药相关的差异表达蛋白,为进一步从多因素角度研究认识维甲酸耐药机制提供了新的线索。     

差异凝胶电泳筛选人骨肉瘤细胞多药耐药相关蛋白

摘 要 目的: 应用差异凝胶电泳分析骨肉瘤多药耐药细胞株与亲本细胞株蛋白质表达的差别，筛查骨肉瘤细胞多药耐药相关蛋白。方法：以小剂量多柔比星（doxorubicin, DXR）诱导人骨肉瘤细胞株MG63，建立骨肉瘤多药耐药细胞株MG63/DXR100。以差异凝胶电泳分离两组细胞中的全部蛋白质，应用图像扫描和DeCyder软件分析差异（表达增加或减少>30%）表达的蛋白质点，对其进行质谱鉴定，并在Mascot 数据库中检索。结果：共检测到明显差异表达的蛋白质点30个，选择其中18个点进行质谱鉴定，有5个蛋白质点鉴定成功，它们分别是与肿瘤细胞转移相关的基质金属蛋白酶1（matrix metalloproteinases 1, MMP1）、具有解毒功能的乙醇脱氢酶6（alcohol dehydrogenase 6, ADH6）、属于抑癌基因的FERM域结合蛋白3（FERM domain containing protein 3, FRMD3），以及两个分别由128和300个氨基酸残基构成的未知蛋白。MMP1、ADH6和2种未知蛋白在耐药细胞表达显著高于非耐药细胞组，而FRMD3表达显著显著低于非耐药细胞。结论：通过差异凝胶电泳筛查到，MMP1、ADH6、FRMD3 及2种未知蛋白质在骨肉瘤细胞的多药耐药细胞和非耐药细胞中差异表达，它们可能参与骨肉瘤细胞的多药耐药机制。     

肺癌骨转移血清蛋白质组学研究

目的:应用蛋白质组学双向荧光差异凝胶电泳和质谱技术筛选肺癌骨转移血清标志.方法:收集肺癌伴骨转移、无骨转移各24例血清样本.同组血清等量混合,用除白蛋白试剂盒除去血清中的白蛋白,再经除盐浓缩后,将4组处理后的血清样品用不同的CyDye染料交叉标记后进行双向荧光差异凝胶电泳(2-D DIGE).利用DeCyder软件分析,将肺癌伴骨转移组与肺癌无骨转移组比较的差异蛋白质点行基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)鉴定.结果:双向荧光差异凝胶电泳显示,肺癌骨转移组血清蛋白中有16个斑点,与肺癌无骨转移组相比差异有统计学意义.其中11个蛋白质表达水平显著增加,选取5个蛋白质表达水平显著下降.5个差异蛋白点进行胶内原位酶解、肽质指纹图谱分析,成功得到4个蛋白质肽质指纹图,并查询数据库初步鉴定该4个蛋白质分别为视黄醇结合蛋白-甲状腺素运载蛋白复合物(RBP-TTR)、结合殊蛋白(Hp)、S-亚硝基血红蛋白(SNO-Hb)和de-sArg141 α Hb.结论:双向荧光差异凝胶电泳结合质谱鉴定是血清差异蛋白质组学研究的可靠平台和有力工具.所鉴定出的蛋白质可能与肺癌骨转移的发生、发展有关,有助于肺癌骨转移发病机制的了解.     

骨肉瘤患者血清蛋白质组学的研究

目的 从蛋白质水平寻找和鉴定骨肉瘤患者血清中的生物学标志物,并初步建立骨肉瘤的血清蛋白质谱.方法 采集8例血清蛋白样本(骨肉瘤患者4例及性别、年龄匹配的正常人对照组4例),用不同的CyDye荧光染料交叉标记后,依次进行双向胶内差异凝胶电泳(2-D DIGE)、图像分析和基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)鉴定.对质谱成功鉴定出的蛋白质,采用Western blot检测方法,进一步验证有意义的差异性蛋白质.结果 对2-D DIGE分析结果进行质谱鉴定,MALDI-TOF-MS成功鉴定出58个蛋白质,经MASCOT等数据库检索证实43个,其中上调18个,下调25个.Western blot检测证实,凝胶溶素(gelsolin)在骨肉瘤血清中低表达.结论 初步寻找到骨肉瘤患者血清中有意义的差异蛋白;gelsolin可能成为骨肉瘤的一个新的血清学诊断标志物,用于患者的大规模筛查,并为将来的基因治疗标定治疗靶点.     

子宫内膜癌耐药细胞B-MD-C1（ADR~（＋/＋））与非耐药细胞B-MD-C1的蛋白质差异研究

目的：比较子宫内膜癌耐药细胞B-MD-C1（ADR~（＋/＋））与非耐药细胞B-MD-C1的蛋白质表达谱,识别和鉴定差异表达蛋白,分析差异蛋白与其耐药的关系。方法：采用双向凝胶电泳及基质辅助激光解吸电离-飞行时间质谱法对B-MD-C1（ADR~（＋/＋））与BMD-C1细胞进行蛋白质分离和表达谱研究,利用生物信息学方法鉴定差异蛋白质。结果：B-MD-C1细胞与B-MD-C1（ADR~（＋/＋））相比,蛋白质表达差异〉2倍的蛋白质点有35个,其中18个蛋白点表达上调,17个蛋白点表达下调。应用Mascot软件查询Swiss-prot数据库初步鉴定差异表达的蛋白质点24个,这些蛋白质可能是B-MD-C1细胞产生耐药的相关蛋白,包括细胞骨架蛋白、代谢酶类、细胞周期相关蛋白、信号转导和凋亡调控相关蛋白、蛋白合成和参与糖酵解的蛋白等。结论：热休克蛋白、β-微管蛋白、丙酮酸激酶同工酶、核糖体蛋白等是B-MD-C1（ADR~（＋/＋））与B-MD-C1细胞间的差异蛋白,这些蛋白多与B-MD-C1（ADR~（＋/＋））细胞耐药密切相关。     

nm23-H1基因转染前后人高转移大细胞肺癌细胞株的比较蛋白质组学研究

背景与目的肿瘤转移抑制基因nm23-H1基因调控肺癌细胞转移潜能的确切机理尚未明了,本研究旨在探讨人高转移大细胞肺癌细胞株(L9981)和转染nm23-H1基因的人高转移大细胞肺癌细胞株(L9981-nm23-H1)的差异表达蛋白,为阐明肺癌转移的分子机制、发现早期诊断肺癌转移的分子标志和新的治疗靶点提供实验依据。方法应用固相pH梯度双向凝胶电泳分离L9981和L9981-nm23-H1细胞株的总蛋白,对25个差异明显的蛋白质点进行质谱鉴定和生物信息学分析。结果研究观察到nm23-H1基因转染使人L9981细胞株蛋白质组的表达谱发生了明显变化:5个蛋白质表达缺失,9个新的蛋白表达,16个蛋白质表达下调,12个蛋白质表达上调。这些蛋白质主要涉及细胞骨架蛋白、信号转导蛋白、细胞代谢相关蛋白、发育增殖相关蛋白及肿瘤侵袭相关蛋白。结论 nm23-H1基因转染L9981后,蛋白质表达谱发生了显著的变化,这些差异蛋白质可能是逆转肺癌侵袭转移的生化基础,本研究结果可能为阐明肺癌转移的分子机制提供线索。     

小细胞肺癌细胞系SBC-5蛋白质组双向凝胶电泳图谱的建立

目的:建立人小细胞肺癌细胞系 SBC-5 蛋白质组双向凝胶电泳图谱.方法:用固相pH梯度双向凝胶电泳技术分离 SBC-5 细胞总蛋白质,凝胶银染显色,PDQuest图像分析系统分析,从凝胶中选取1个分离较好的蛋白质点,应用基质辅助激光解析离子化-飞行时间-质谱技术(MALDI-TOF-MS)和数据库搜索鉴定蛋白质.结果:获得了背景清晰、分辨率和重复性较好的双向凝胶电泳图谱,三块胶平均蛋白质点数为1116±64,匹配率达84%.蛋白质点分布以 PI 4-7、相对分子质量20 000-80 000范围内最多.1个蛋白点通过质谱分析和数据库检索得到了初步的鉴定.结论:建立了人小细胞肺癌细胞系SBC-5蛋白质组双向电泳参考图谱,为其蛋白质组学的进一步研究奠定了基础,也为人小细胞肺癌蛋白质组表达数据库的建立提供了有意义的数据.     

应用蛋白芯片技术筛选肺腺癌化疗敏感血清蛋白标志物

目的 探讨肺腺癌化疗敏感患者与化疗耐药患者血清蛋白质质谱的不同,筛选肺腺癌化疗敏感血清蛋白标志物。方法 用WCX2蛋白芯片结合表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术,检测22例肺腺癌化疗敏感的患者和21例化疗不敏感的血清蛋白质谱,筛选出差异表达蛋白质。结果 发现利用M/Z(质荷比)为16083.50Da,7963.17Da,16121.50Da,15921.60Da,8066.79Da,8040.17Da和3394.57Da的7个差异蛋白可区分标准含铂类方案化疗敏感组和耐药组,联合m/z为7963.17Da与16083.50Da的差异蛋白建立预测模型,可预测肺腺癌标准含铂类方案化疗敏感/耐药性,其敏感性86.36%和特异性80.95%。结论 SELDI-TOF-MS技术是寻找肺腺癌化疗敏感血清诊断标志物的有效工具。     

EGFR基因敏感突变晚期肺腺癌一线培美曲塞同药维持化疗疗效分析

目的:分析表皮生长因子受体(EGFR)基因敏感突变晚期肺腺癌患者一线培美曲塞维持化疗疗效。方法:对我院2012年6月始至今收治的伴有EGFR基因19或21外显子敏感突变的IIIb-IV期肺腺癌患者,一线接受培美曲塞联合顺铂后同药维持化疗的无进展生存时间(PFS)进行回顾性分析。结果:10例EGFR基因19和/或21外显子敏感突变的IIIb-IV期肺腺癌患者一线接受培美曲塞联合顺铂同药维持治疗,完全缓解(CR) 0例,部分缓解（PR）6例（60%）,稳定（SD）4例（40%）,疾病控制率（DCR）10例（100%）,中位无进展生存时间（PFS）12.0个月（95%可信区间:7.60~16.30）,1年生存率100%。主要毒副反应为骨髓抑制、恶心呕吐、疲劳乏力、神经毒性,安全可耐受。结论:EGFR基因敏感突变晚期肺腺癌患者一线培美曲塞维持化疗可取得较好疗效。     

Combination chemotherapy of oxaliplatin and 5-fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ovary: A potential treatment strategy

Resistance of ovarian mucinous adenocarcinoma to standard chemotherapy with paclitaxel and carboplatin is associated with poor prognosis, and an effective treatment is needed. The present study aimed to identify an effective chemotherapy for ovarian mucinous adenocarcinoma. Five human ovarian mucinous adenocarcinoma cell lines (MN-1, OMC-1, RMUG-L, RMUG-S, TU-OM-1) were used in this study. Sensitivity of the cells to the anticancer agents was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and we assessed drug sensitivity by calculating the assay area under the curve for each agent. Protein expression was confirmed by Western blot analysis. We also examined the efficacy of combination chemotherapy on survival in a xenograft model of nude mice. The IC₅₀ to anticancer agents ranged widely. The assay area under the curve indicated that two of five cell lines (MN-1, TU-OM-1) were sensitive to oxaliplatin, 5-fluorouracil and etoposide, and only one (TU-OM-1) was sensitive to 7-ethyl-10-hydroxycamptothecin, which is an active metabolite of camptothecin. All cell lines were resistant to cisplatin and paclitaxel. The combination of oxaliplatin and 5-fluorouracil resulted in additive or synergistic effects on all cell lines. The combination of oxaliplatin and 5-fluorouracil significantly prolonged survival in a ovarian mucinous adenocarcinoma xenograft model of nude mice. Protein expression levels of the excision repair cross-complementation group 1 were lower in oxaliplatin sensitive cell lines. Exposure to 5-fluorouracil down-regulated cross-complementation group 1 expression in ovarian mucinous adenocarcinoma cells. We conclude that combination chemotherapy consisting of oxaliplatin and 5-fluorouracil was an effective treatment for ovarian mucinous adenocarcinoma and may be a pivotal candidate for a novel treatment strategy. ( Cancer Sci 2009; 100: 546–551)     

The expression and significance of the enhancer of zeste homolog 2 in lung adenocarcinoma

Lung adenocarcinoma, with increased incidence in the world, exhibits poor prognosis and is usually resistant to conventional chemotherapy due to drug resistance. The enhancer of zeste homologue 2 (EZH2), a histone methyltransferase, plays a key role in tumorigenesis and cancer development through chromatin remodeling in various types of cancer. However, its potential role in lung adenocarcinoma has not been well defined. In this study, the expression of EZH2 was examined in lung adenocarcinoma tissues and cell lines. Most interestingly, EZH2 overexpression was detected in tumor tissue and significantly correlated with histological differentiation, pathological tumor-node-metastasis stage and smoking history, but not with gender or age. Furthermore, EZH2 overexpression was also detected in cisplatin-resistant cancer cells rather than cisplatin-sensitive cells. Short hairpin RNA targeted against EZH2 inhibited cell proliferation and migration, and led to G(2)/M cell cycle arrest and apoptosis in both cisplatin-resistant and cisplatin-sensitive cell lines. Moreover, EZH2 knockdown enhanced cisplatin sensitivity of cisplatin-resistant cells and reduced the expression levels of multidrug resistance-related protein 1. Our study suggests that EZH2 contributes to the progression of lung adenocarcinoma, and the deletion of EZH2 inhibits cancer and resensitizes cells to cisplatin in lung adenocarcinoma.     

应用MALDI-TOF-MS检测肺鳞癌患者血清多肽并分析其与化疗疗效相关性

背景与目的 晚期肺鳞癌(squamous cell carcinoma of lung,SCC)一线治疗以化疗为主,其标准铂二联方案化疗只能给患者带来有限的获益.并且不同的患者对于化疗药物的获益不同.所以实现化疗药物最优选择达到个体化预见性治疗尤为重要.本研究应用基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorp-tion/ionization-time of flight-mass spectrometry,MALDI-TOF-MS)检测初治晚期SCC患者接受紫杉醇类联合铂类化疗前血清多肽,并分析其与化疗疗效的相关性.方法 初治晚期SCC患者接受紫杉醇类联合铂类方案化疗,每两周期进行疗效评价.评效为完全缓解(complete response,CR)或部分缓解(partial response,PR)患者定义为化疗敏感组,疾病进展(progressive disease,PD)患者定义为耐药组.留取SCC患者化疗前血清样本,81例患者按照3:1的比例随机分为训练组(敏感组I与耐药组I)和验证组(敏感组II与耐药组II),预处理训练组血清样本并进行MALDI-TOF-MS检测,得到血清多肽指纹图谱.经ClinProTools软件系统分析处理,得到敏感组I与耐药组I的差异多肽.应用软件内置的3种不同的生物学算法分别建立疗效预测模型,选取最优算法建立疗效预测模型.运用验证组进行盲样验证.结果 训练组共纳入30例敏感组患者,31例耐药组患者;验证组共纳入敏感与耐药组患者各10例.训练组在敏感与耐药组有96个差异多肽,其中具有统计学意义的多肽有16个(P<0.001).由5个多肽(1,897.75 Da,2,023.93 Da,3,683.36 Da,4,269.56 Da,5,341.29 Da)建立疗效预测模型.该模型对化疗敏感组患者的识别率为95.11%,交叉验证率为89.18%.经验证组进行盲样验证,其模型的准确率为85%,灵敏度为90.0%,特异性为80.0%.敏感组I中位无进展生存期(progress free survival,PFS)为7.2个月(95%CI:4.4-14.5);耐药组I中位PFS为1.8个月(95%CI:0.7-3.5).结果发现:4,232.04 Da、4,269.56 Da的差异多肽与SCC患者PFS存在相关性(P<0.001).结论 应用MALDI-TOF-MS技术可检测到化疗敏感组及耐药组患者的血清多肽存在差异,初步建立的疗效预测模型可用于预测紫杉醇类联合铂类方案化疗疗效.但需进一步扩大样本量完善及验证模型.     

A case of advanced clear cell carcinoma of the endometrium that responded remarkably to neoadjuvant chemotherapy of combination carboplatin plus weekly paclitaxel

Clear cell carcinoma of the endometrium is a very rare and highly malignant neoplasm that accounts for less than 5% of endometrial carcinoma. Survival of patients in the advanced stage is poor, and the treatment of choice is not clear. We report the case of a 62-year-old woman who had Stage IVb advanced clear cell carcinoma of the endometrium with multiple lung metastases. The lesions were considered surgically incurable, so she was placed on neoadjuvant chemotherapy of combination carboplatin (CBDCA) (AUC 5, day 1) plus weekly paclitaxel (PTX) (70 mg/m(2), day 1, 8, 15). After 3 courses of chemotherapy, the uterine tumor was obviously reduced, and lung metastases had disappeared. Therefore, she underwent the operation. The current case suggests that combination CBDCA plus weekly PTX is effective against advanced clear cell carcinoma of the endometrium.     

An early event of EGFR mutation in pleomorphic carcinoma of the lung

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective in prolonging progression-free survival time of patients with non-small cell lung carcinoma, typically adenocarcinoma, bearing some active EGFR mutations in their tumors. However, the close relationship between the EGFR mutations and pleomorphic carcinoma of the lung, which is a very rare type of primary lung cancer, has never been elucidated. We present a 60-year-old Japanese woman with pleomorphic carcinoma of the lung that became resistant to cytotoxic chemotherapies including platinum-based chemotherapy, and her general condition seriously deteriorated. Thereafter, treatment with gefitinib was started and resulted in significant tumor shrinkage and a dramatic improvement in her general condition for up to 8.5 months. Analyses of the EGFR mutation in separately microdissected specimens from adenocarcinoma and spindle cell components revealed that both components possessed the L858R point mutation. These findings gave us some insight into the carcinogenesis of pleomorphic carcinoma of the lung in relation to EGFR gene alteration. Testing for EGFR mutation may be important in patients with advanced pleomorphic carcinoma including adenocarcinoma component that is usually chemoresistant.     

A case of gastric cancer with lung, Virchow and para-aortic lymph node metastases treated successfully using PTX/5'-DFUR

A 6 3-year-old male presented with dysphagia. Gastrointestinal endoscopic examination showed advanced gastric cancer type 3, which was diagnosed as well-differentiated adenocarcinoma. Computed tomography(CT)showed bilateral lung tumors, hugely enlarged Virchow and para-aortic lymph nodes. He was treated with combination chemotherapy of weekly paclitaxel(PTX)and doxifluridine(5'-DFUR). PTX was administered at a dose of 80mg/m2 on day 1, 8 and 15, and 5'- DFUR was orally administered at a dose of 533mg/m / 2day for 5 days followed by withdrawal for 2 days. After four courses of treatment, CT showed an almost complete disappearance of the lung and lymph node metastases. After 13 courses of treatment, total gastrectomy and lymph node dissection were performed. One year postoperatively, the patient died of a recur- rence.     

紫杉醇加顺铂治疗晚期非小细胞肺癌的临床疗效

目的 观察紫杉醇(PTX)加顺铂(DDP)联合化疗治疗晚期非小细胞肺癌的近期疗效。方法 PTX135～180mg／m^2静脉滴注，第1天；DDP40mg／次，静脉滴注，第1～3天。每21天为一个周期，连用2或3周期。结果 可评价疗效者46例．其中CR1例(2．2％)，PR16例(34．8％)，总有效率为37．0％。毒副反应主要是骨髓抑制和恶心呕吐。结论 PTX DDP治疗晚期非小细胞肺癌可获得较高疗效，毒副反应轻，是个较好的联合化疗方案。     

肺鳞癌的个体化治疗进展

鳞状细胞肺癌(SqCLC)是非小细胞肺癌(non-small cell lung cancer，NSCLC)的一种独特的组织学亚型，尤其是晚期鳞癌，由于其特殊的临床病理特征，相对于肺腺癌可选择的治疗药物少，不良反应也大，因此肺鳞癌预后比肺腺癌差。近年来，随着化疗药物的改进，靶向药物的研发，尤其是免疫治疗的重大突破，治疗肺鳞癌有了更多的选择，肺鳞癌也走向了个体化治疗。