宫颈腺癌发生相关蛋白的蛋白质组学初步研究

目的：为了研究宫颈腺癌和正常宫颈组织的差异表达蛋白,为宫颈腺癌的发生和早期诊断提供有意义的生物标志物。方法：以正常宫颈组织和宫颈腺癌组织为研究对象,提取组织总蛋白,依次进行二维凝胶电泳,凝胶图象分析,基质辅助激光解吸附／离子化飞行时间质谱及生物信息学分析。Western Blot方法验证部分蛋白表达情况。结果：建立了宫颈腺癌和正常宫颈组织的二维电泳图谱,进行质谱和生物信息学分析比较鉴定宫颈腺癌和正常宫颈组织差异表达蛋白7个,与正常宫颈组织比较,宫颈腺癌表达降低的蛋白有3个,包括抑制素（inhibin-beta）,PTEN,乳铁蛋白（lactoferrin）;宫颈腺癌表达升高的蛋白有4个,包括谷胱甘肽S-转移酶（GSTT1＊0）,Homeodomain—interacting protein kinase2（HIPK2）,CD44v5,galectin-7。Western Blot方法检测结果显示inhibin-beta和PTEN在宫颈腺癌中表达变化情况与蛋白质组学结果一致。结论：宫颈腺癌和正常宫颈组织存在差异表达蛋白,这些差异表达蛋白可能是宫颈腺癌发生相关蛋白,可能作为宫颈腺癌早期诊断的标志物。     

临床蛋白质组学———蛋白质组学在临床研究中的应用

临床蛋白质组学是将蛋白质组学技术应用于临床医学研究,它主要围绕疾病的预防、早期诊断和治疗等方面开展研究,其中,恶性肿瘤是临床蛋白质组学研究的一个重点研究对象.由于肿瘤生物标志物对早期诊断具有重要价值,所以临床蛋白质组学的主要目标之一是寻找合适的肿瘤生物标志物,多分子生物标志物已成为寻找肿瘤生物标志物的一个研究趋势.简要介绍了临床蛋白质组学的基本概念,实验设计,临床样本收集与预处理以及蛋白质组学技术在临床研究中的应用与进展.     

应用蛋白质组学技术筛选胃癌SGC-7901细胞中的let-7a功能相关蛋白

为探讨let-7a表达下调在胃癌发病中的机制,高通量地检测了与let-7a功能相关的蛋白质.首先采用基因克隆技术稳定过表达SGC-7901细胞系的let-7a基因,然后用蛋白质组学技术研究稳定过表达该基因对SGC-7901细胞蛋白质表达谱的影响.通过对SGC-7901/let-7a细胞的蛋白质表达谱改变的研究,并用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析鉴定了10个差异表达蛋白质.这些差异表达蛋白质可能是let-7a功能相关蛋白质,其中抗氧化蛋白2、胰岛素样生长因子结合蛋白2、二硫化蛋白异构酶A2、四氢叶酸合成酶、细胞周期素依赖性激酶抑制蛋白1、Rho-GTPsae激活蛋白4表达上调,Skp2蛋白、血小板黏附蛋白CD41、纤维连接蛋白、Cks1蛋白表达下调.部分差异表达蛋白质如细胞周期素依赖性激酶抑制蛋白1、Skp2蛋白和纤维连接蛋白经蛋白质印迹分析进行了验证.在SGC-7901/let-7a中鉴定的10个差异表达蛋白质涉及到细胞周期的调控、分子基因表达调控、细胞黏附、细胞代谢等众多事件,它们可能作为let-7a功能相关蛋白质,为阐明let-7a表达下调在胃癌发病中的机制提供了重要线索.     

应用蛋白质组学筛选宫颈癌患者尿液中的肿瘤标志物

通过比较健康女性和宫颈癌患者的尿蛋白质组,发现并分析差异表达蛋白,从中筛选潜在的宫颈癌的标志物。研究对象由43名宫颈癌患者（CC）和47名健康女性（HW）组成。用超速离心法沉淀尿蛋白,再用一维凝胶电泳（SDS-PAGE）与液相色谱-质谱联用技术（LC-MS/MS）鉴定尿液中的蛋白质,蛋白质定量采用无标定量。比较患者尿蛋白质组、健康对照的尿蛋白质组和宫颈癌组织蛋白质组,有1910个蛋白质是患者和健康对照共有的尿蛋白,这其中有746个蛋白质也存在于宫颈癌组织蛋白质组。在这746个蛋白质中找到84个上调蛋白和82下调蛋白。通过生物信息学分析发现牛皮癣素（S100A7）和癌胚抗原相关细胞黏附分子8（CEACAM8）是宫颈癌尿液样本独有蛋白质。在验证组的70例样本中,双盲法测试S100A7、CEACAM8以及两者联合诊断宫颈癌的敏感性能达到73%、87%、93%。结果提示,宫颈癌患者的尿蛋白质组与健康女性的尿蛋白质组不同,并且S100A7和CEACAM8可以作为宫颈癌潜在的肿瘤标志物。     

CT和MRI在宫颈癌分期诊断中的临床对比分析

目的:比较CT和MRI在宫颈癌分期诊断中的临床应用价值。方法:回顾性分析宫颈癌患者96例,上述患者均有完整临床资料及术前CT和MRI检查结果,根据术后病理检查结果,比较两种检查方法影像学诊断、侵犯宫颈旁组织、侵犯子宫体、侵犯阴道、淋巴结转移的术前检查结果与病理诊断的符合率。结果:两种检查方法在宫颈癌Ⅰb期、Ⅱa期和Ⅱb期诊断符合率比较差异具有高度统计学意义(P<0.01);宫颈癌Ⅲa期和Ⅲb期符合率比较,差异具有统计学意义(P<0.05);宫颈癌Ⅳ期符合率比较,差异无统计学意义(P>0.05);检查提示侵犯宫颈旁组织、侵犯子宫体与侵犯阴道与术后病理诊断符合率比较,差异具有统计学意义(P<0.05)。结论:对于宫颈癌患者,术前应积极采取MRI检查方式,可提高诊断符合率,有利于对患者术后治疗的开展和预后的判断。     

CT和MRI在宫颈癌分期诊断中的临床对比分析

目的：比较CT和MRI在宫颈癌分期诊断中的临床应用价值。方法：回顾性分析宫颈癌患者96例,上述患者均有完整临床资料及术前CT和MRI检查结果,根据术后病理检查结果,比较两种检查方法影像学诊断、侵犯宫颈旁组织、侵犯子宫体、侵犯阴道、淋巴结转移的术前检查结果与病理诊断的符合率。结果：两种检查方法在宫颈癌Ⅰb期、Ⅱa期和Ⅱb期诊断符合率比较差异具有高度统计学意义（P〈0．01）;宫颈癌Ⅲa期和Ⅲb期符合率比较,差异具有统计学意义（P〈0．05）;宫颈癌Ⅳ期符合率比较,差异无统计学意义（P〉O．05）;检查提示侵犯宫颈旁组织、侵犯子宫体与侵犯阴道与术后病理诊断符合率比较,差异具有统计学意义（P〈0．05）。结论：对于宫颈癌患者,术前应积极采取MRI检查方式,可提高诊断符合率,有利于对患者术后治疗的开展和预后的判断。     

蛋白质组学的相关技术及应用

当今分子生物学领域内,蛋白质组已成为研究的热点。基因组相对较稳定,而且各种细胞或生物体的基因组结构有许多基本相似的特征;蛋白质组是动态的,随内外界刺激而变化。对蛋白质组的研究可以使我们更容易接近对生命过程的认识。但同时对数千种(甚至更多)蛋白质特性的研究也是一个很大的技术挑战。双相凝肢电泳、质谱、酵母双杂交技术以及生物信息学的发展在一定程度上解决了这一技术难题。本对此类技术及其在各领域的应用作一简要介绍。     

蛋白质组学技术在精子蛋白研究中的应用

从蛋白质组学研究的技术手段、蛋白质组学在人类不育及精卵相互识别并结合的机理研究、免疫法开展男性避孕方法的研究及蛋白质组学研究方法在家畜繁殖环节中的应用等几个方面阐述了蛋白质组学在人类生殖及动物繁殖环节相关研究中的重要作用。说明蛋白质组学已经成为生命科学未来发展的主要分支之一,为揭示生命个体的蛋白质动态变化提供了技术手段和理论基础,并将在药物开发,生命活动机理研究等方面发挥巨大作用,也必将会在家畜繁殖学领域发挥其应有的作用。     

差异蛋白质组学在筛选肿瘤标记中的应用

目前,利用差异蛋白质组技术对肿瘤早期标记蛋白的筛选已在世界范围内形成热潮。介绍了蛋白组学、差异蛋白质组学、肿瘤标记、常用的差异蛋白质组学技术及其在筛选肿瘤标记中的应用．最后指出了研究中存在的问题,并对前景进行了展望。     

应用蛋白质组学和RNAi技术筛选鼻咽癌细胞中p53功能相关蛋白质

为了阐明鼻咽癌中高表达的p53蛋白聚集与失活的机制,高通量地检测与p53功能相关的蛋白质,首先采用RNA干扰(RNAi)技术稳定沉默鼻咽癌细胞系CNE2的p53基因表达,然后用蛋白质组技术研究稳定沉默该基因对鼻咽癌蛋白质表达谱的影响.通过对稳定干扰p53基因后鼻咽癌细胞系CNE2的蛋白质表达谱改变的研究,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析和电喷雾串联质谱(ESI-Q-TOF-MS)验证鉴定了22个差异表达蛋白质.在这些差异表达蛋白质中,有些是已经报道的p53功能相关蛋白质,如热休克蛋白27(HSP27)、异质性胞核核糖核蛋白K(hnRNPK)、14-3-3σ等,其他可能是新的p53功能相关蛋白质,如eIF4B、TPT1、hnRNPH3、SFRS1等.部分差异表达蛋白质如HSP27、14-3-3σ和GRP75经蛋白质印迹分析技术进行了验证,同时pcDNA3.1-FLAG-p53质粒转染CNE2细胞引起了HSP27、14-3-3σ表达下调,GRP75表达上调.在鼻咽癌细胞中鉴定的22个差异表达蛋白质大致可以分为5类,包括信号传导相关蛋白质、分子伴侣、与转录和翻译相关蛋白质、代谢相关蛋白和细胞结构相关蛋白质,涉及到细胞周期的调控、分子基因表达调控、细胞黏附、细胞代谢等众多事件,它们可能作为p53功能相关蛋白质,为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.     

Proteomic identification of molecular targets of gambogic acid: Role of stathmin in hepatocellular carcinoma

Gamboge has been developed as an injectable drug for cancer treatment in China. In this study, the inhibition ratio and their IC₅₀ values of two derivatives from Gamboge in hepatocellular carcinoma (HCC) were determined. Proteomic approach was employed to reveal the target proteins of these two derivatives, gambogic acid (GA), and gambogenic acid (GEA). HCC cells were cultured under varied conditions with the addition of either GA or GEA. Twenty differentially expressed proteins were identified and the four most distinctly expressed proteins were further validated by Western blotting. GA and GEA revealed inhibitory effects on HCC cell proliferation. The expression of cyclin‐dependent kinase 4 inhibitor A and guanine nucleotide‐binding protein β subunit 1 were upregulated by both xanthones, whilst the expression of 14‐3‐3 protein sigma and stathmin 1 (STMN1) were downregulated. Furthermore, overexpression of STMN1 in HCC cells decreased their sensitivity, whilst small interfering RNAs targeting STMN1 enhanced their sensitivity to GA and GEA. In conclusion, our study suggested for the first time that STMN1 might be a major target for GA and GEA in combating HCC. Further investigation may lead to a new generation of anticancer drugs exerting synergistic effect with conventional therapy, thus to promote treatment efficacy.     

Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinoma

Enhancer of zeste homolog 2 (EZH2) is suggested to be a potential therapeutic target and a diagnostic marker for cancer. Our previous study also showed the critical role of EZH2 in hepatocellular carcinoma (HCC) tumorigenesis. The present study is aimed at revealing the comprehensive downstream pathways of EZH2 by functional proteomic profiling. Lentivirus mediated RNA interference (RNAi) was employed to knockdown EZH2 in HCC cells. The 2-DE was employed to compare the expression profile difference between parental and EZH2-knockdown HCC cells. In total, 28 spots were differentially expressed during EZH2 inhibition. Among all, 18 proteins were identified by PMF with MALDI-TOF MS. Western blotting further validated upregulation of 60S acidic ribosomal protein P0 (L10E), and downregulation of two proteins with EZH2 inhibition: stathmin1 and probable protein disulfide isomerase (PDI) ER-60 precursor (ERp57). Moreover, L10E was downregulated with overexpression of EZH2 in hepatocytes, and L10E reversed the effect of EZH2 on cell proliferation, suggesting it a downstream target of EZH2. The comprehensive and comparative analyses of proteins associated with EZH2 could further our understanding on the downstream signal cascade of EZH2 leading to tumorigenesis.     

CMTM5-v1 induces apoptosis in cervical carcinoma cells

CMTM5 (CKLF-like MARVEL transmembrane domain-containing member 5) exhibits tumor inhibition activity with frequent epigenetic inactivation in various tumor cell lines including cervical carcinoma (CC) cells. In this paper, we examined the function of CMTM5-v1 (the primary RNA splicing form) in both HeLa and SiHa cells. Overexpression of CMTM5-v1 in both cells can induce apoptosis, but the effects are more obvious in SiHa than that in HeLa. In SiHa cells, restoration of CMTM5-v1 caused disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase3 and cleavage of PARP. General caspase inhibitor almost prevented apoptosis of SiHa cells, suggesting that CMTM5-v1 induces apoptosis mainly through caspase-dependent pathway. These findings verify that CMTM5-v1 inhibits the growth of CC cell lines via inducing apoptosis.     

无核荔枝胚胎发育时期蛋白质图谱分析

通过二维聚丙烯酰胺凝胶电泳(2DE)以及计算机辅助的图像分析技术,对荔枝开花后20d的正常与败育胚蛋白质图谱进行了初步分析。结果表明,正常胚总蛋白质斑点数为129,败育胚总蛋白质斑点数为130,其中24个蛋白质点在两种胚中的表达丰度没有明显变化,35个蛋白质点在表达丰度上有明显差异,55%的蛋白则发生了蛋白质缺失、增加以及位置改变等变化。这两种蛋白质组的表达差异说明了胚内蛋白质成分在其败育过程中发生了变化,这些蛋白可能参与了胚败育的调节和控制。     

Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo

A recent report showed that reversine treatment could induce murine myoblasts dedifferentiation into multipotent progenitor cells and inhibit proliferation of some tumors, and other reports showed that apoptosis of lung adenocarcinoma cells could be induced by aspirin. The aim of the present study was to evaluate the synergistic antitumor effects of reversine and aspirin on cervical cancer. The inhibition rate of reversine and aspirin on cervical cancer cell lines’ (HeLa and U14) was determined by MTT method, cell cycle of HeLa and U14 cells was analyzed by FACS, mitochondrial membrane potential of HeLa and U14 was detected using a JC-1 kit. HeLa and U14 colony formation was analyzed by soft agar colony formation assay. The expression of caspase-3, Bcl-2/Bax, cyclin D1 and p21 was detected by qRT-PCR and Western Blotting. Moreover, tumor weight and tumor volume was assessed using a murine model of cervical cancer with U14 cells subcutaneously (s.c.) administered into the neck, separately or combined with drug administration via the intraperitoneal (i.p.) route. The inhibition rate of cells in the combination group (10 μmol/L reversine, 10 mmol/L aspirin) increased significantly in comparison to that when the drugs were used alone (P < 0.05); moreover, this combination could synergistically inhibit the proliferation of five cervical cancer cell lines (HeLa, U14, Siha, Caski and C33A). In the therapeutic mouse model, tumor weight and tumor volume of cervical cancer bearing mice was more reduced when compared with the control agents (P < 0.05) in tumor-bearing mice. The combination of reversine and aspirin exerts synergistic growth inhibition and apoptosis induction on cervical cancers cells.     

Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins

Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, we have used a systems biology approach to study the molecular basis of aging of the mouse heart. We have identified 8 protein spots whose expression is up-regulated due to aging and 36 protein spots whose expression is down-regulated due to aging (p0.05 as judged by Wilcoxon Rank Sum test). Among the up-regulated proteins, we have characterized 5 protein spots and 2 of them, containing 3 different enzymes, are mitochondrial proteins. Among the down-regulated proteins, we have characterized 27 protein spots and 16 of them are mitochondrial proteins. Mitochondrial damage is believed to be a key factor in the aging process. Our current study provides molecular evidence at the level of the proteome for the alteration of structural and functional parameters of the mitochondria that contribute to impaired activity of the mouse heart due to aging.     

宫颈疾病与宫颈微生态特征的研究进展

宫颈微生态是一个复杂的生态系统,可随年龄、月经周期、妊娠、外源性因素等原因而发生动态变化,这种平衡对于机体健康至关重要。宫颈微生态包括菌群、局部pH、内分泌调节和免疫系统等,其中大量定植的宫颈菌群是微生态体系的核心组成部分。当发生感染、局部pH改变、雌激素减退等情况时,菌群生态平衡可被破坏,致病菌可过量繁殖成为优势菌,同时提升人乳头瘤病毒(HPV)感染风险,引起宫颈炎、宫颈鳞状上皮化生、宫颈癌等等。研究认为,宫颈微生态的改变与HPV感染、宫颈疾病的发生具有密切的相关性。然而国内关于宫颈微生态与宫颈疾病的直接关系的报道尚不多见,本文就宫颈微生态失衡与其引起的常见宫颈疾病,如宫颈炎、宫颈上皮内瘤变、宫颈癌等之间的相关性进行探讨。