放射性肝损伤的血清比较蛋白质组学分析

目的: 通过分离、鉴定及验证SD肝硬化大鼠肝脏放疗前后血清差异蛋白质的表达,探讨可能用于检测及监控放射性肝损伤的早期生物学指标。方法: 8只健康成年SD大鼠皮下注射四氯化碳(CCl₄),连续6周,建立肝硬化模型后随机分组,每组4只,即对照组和实验组。对照组大鼠(CCl₄组)未给予放疗,实验组大鼠(CCl₄+放疗组)行半肝照射,单次剂量15 Gy。6 h后分别提取2组大鼠的血清总蛋白,进行比较蛋白质组学分析。采用双向凝胶电泳分离蛋白,经图像分析识别2组大鼠放疗前后差异表达的蛋白质。应用基质辅助激光解析电离飞行时间质谱(MALD-TOF-MS)鉴定差异蛋白质。应用Western blotting对血清中乙酰肝素酶和肝细胞生长因子受体的表达水平进行验证。结果: 获得了分辨率和重复性均较好的凝胶蛋白图谱。共筛选出33个在实验组明显差异表达的蛋白点,其中12个蛋白质鉴定成功。在这12个蛋白质中,实验组中5个表达上调,7个表达下调。Western blotting进一步证实了实验组乙酰肝素酶表达上调,肝细胞生长因子受体表达下调。结论: 成功鉴定了12个与放射性肝损伤相关的蛋白。乙酰肝素酶和肝细胞生长因子受体可能作为预测及监控放射性肝损伤的早期指标。临床上要将二者作为可能的生物学标志物,其敏感性和特异性还有待进一步研究。     

急性心肌缺氧对乳鼠心肌细胞脑钠尿肽表达的影响及其作用机制

目的: 观察急性缺氧损伤对乳鼠心肌细胞脑钠尿肽(BNP)表达水平的影响并探讨其可能作用机制。方法: 原代乳鼠心肌细胞缺氧、无糖、无血清培养以模拟心肌缺血损伤,利用CCK-8法测细胞存活率、ELISA法测白细胞介素-6(IL-6)和BNP的表达;以IL-6直接干预体外培养的心肌细胞, 采用RT-PCR、Western blotting和ELISA方法观察BNP、转化生长因子β₁(TGF-β₁)、Smad2 mRNA的转录和蛋白的表达。结果: 缺氧显著上调IL-6和BNP的表达,且两者呈线性正相关;IL-6直接干预可剂量和时间依赖性地上调心肌细胞BNP的mRNA和蛋白表达水平,同时TGF-β₁和Smad2的表达水平亦增加;而针对TGF-β₁的中和抗体能够部分地抑制由IL-6引起的BNP表达增加。结论: 急性心肌缺氧可直接上调BNP的表达水平,这种调节效应与TGF-β₁/Smad2信号通路上调有关。     

O3抑制Notch信号通路减少OA大鼠软骨细胞凋亡的实验研究

目的 观察大鼠骨关节炎（OA）软骨细胞中Notch信号通路相关因子（Notch-1、Jagged1、keratin1、involucrin）及凋亡生物标志物（caspase-3、Bax、Bcl-2）的表达情况,探索臭氧（O₃）作用于OA的具体机制。方法 选取SD大鼠24只,按随机数字表分为空白组、模型组、模型+O₃治疗组,每组8只。使用木瓜蛋白酶合半胱氨酸混合液关节腔注射法复制OA模型（右膝）,造模成功后,每周在模型+O₃治疗组大鼠右膝关节腔内注入2 mL O₃（25 μg/mL）1次,模型组注入等体积空气,空白组不做处理;治疗4周后处死大鼠,收集膝OA关节软骨组织,行HE染色观察软骨组织,免疫荧光检测caspase-3、Notch-1表达情况,Western Blot检测Notch-1、Jagged1、Bcl-2、keratin1、involucrin的表达情况。结果 HE染色观察显示,模型+O₃治疗组能有效改变软骨退变,使软骨细胞成簇增生,潮线间断。免疫组化及Western Blot检测显示,与空白组相比,caspase-3、Notch-1、Jagged1和Bax蛋白含量在模型组中显著增高（P＜0.05）;模型+O₃治疗组与模型组比较,上述蛋白含量均有所下降,其中caspase-3和Notch-1蛋白含量与模型组相比下降程度更显著,差异有统计学意义（P＜0.05）;而Bcl-2、keratin1和involucrin蛋白含量在模型组显著降低（P＜0.05）,经O₃治疗后上述蛋白含量显著增高（P＜0.05）。结论 Notch信号通路通过调控OA软骨细胞凋亡参与OA发生发展的过程,而O₃能通过抑制Notch信号通路减少软骨细胞凋亡,保护关节软骨,延缓OA进展。     

远志皂苷元对H2O2诱导的大鼠海马神经元损伤的影响及其机制

目的: 观察远志皂苷元(senegenin, Sen)对过氧化氢(H₂O₂)诱导的SD大鼠海马神经元损伤的影响并探讨其作用机制。方法: SD大鼠海马神经元培养至第6 d,随机分为正常组、H₂O₂组、Sen+ H₂O₂ 组和Sen组,药物干预后检测细胞存活率、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,并用 Hoechst 33258染色观察细胞核形态变化,荧光定量PCR(real-time PCR)检测神经元凋亡相关基因 bcl-2和bax 的表达,进一步采用Western blotting观察Bcl-2和Bax蛋白表达,酶荧光活性检测试剂盒测定caspase-3的活性改变。结果: 与正常组相比,H₂O₂组细胞存活率降低。与H₂O₂组相比,20 μmol/L Sen+ H₂O₂ 组细胞存活率升高,SOD活性升高,MDA含量下降,并且远志皂苷元能够上调bcl-2 mRNA表达、下调bax mRNA表达,降低caspase-3活性,Western blotting结果进一步表明Bcl-2蛋白表达升高,Bax蛋白表达下降,各指标均有显著差异(P<0.05)。结论: 远志皂苷元对H₂O₂处理的海马神经元有一定保护作用,其机制可能与远志皂苷元增强海马神经元抗氧化能力、调节细胞凋亡相关蛋白从而抑制凋亡有关。     

Bmi-1 基因过表达对人正常胃黏膜上皮细胞株GES-1增殖的影响

目的: 探讨原癌基因B淋巴瘤莫洛尼鼠白血病病毒插入区1( Bmi-1 )过表达对人正常胃黏膜上皮细胞株GES-1增殖的影响。方法: 采用逆转录病毒介导转染方法将携带原癌基因 Bmi-1 的质粒或空质粒稳定转染GES-1细胞,通过real-time PCR及Western blotting在mRNA及蛋白水平鉴定转染效果。流式细胞术检测过表达 Bmi-1 对GES-1细胞周期的影响。应用CCK-8(Cell Counting Kit-8)试剂盒检测稳定转染 Bmi-1 对GES-1细胞增殖的影响。结果: Real-time PCR及Western blotting结果均表明成功建立稳定转染 Bmi-1 基因的GES-1细胞株。流式细胞术结果表明,过表达 Bmi-1 基因使GES-1细胞G₀/G₁期减少,G₂/M期和S期细胞增多。生长曲线显示,过表达 Bmi-1 基因使GES-1细胞增殖速度明显提高。结论: 过表达 Bmi-1 基因能调控GES-1细胞的细胞周期,促进GES-1细胞的增殖。     

p38信号通路对SETD4在细胞中的表达和定位的影响

目的: 观察SETD4(SET domain containing 4)蛋白在不同细胞中的表达和定位情况,以及亚砷酸钠(NaAsO₂)刺激对细胞SETD4表达和定位的影响,探讨NaAsO₂诱导SETD4的改变是否依赖于p38信号通路。方法: 用Western blotting检测SETD4在不同细胞中蛋白的表达情况,用细胞免疫荧光技术检测SETD4在细胞中的定位;用NaAsO₂刺激 p38 ^+/+和 p38 ^-/-细胞,分别收集细胞总蛋白检测SETD4蛋白的表达变化,同时观察SETD4的定位和移位改变。结果: SETD4蛋白无论在鼠源还是人源的细胞中均有表达;细胞免疫荧光结果显示SETD4在整个细胞中分布,以胞质较多; p38 ^+/+和 p38 ^-/-细胞受NaAsO₂刺激后其SETD4蛋白的表达趋势一致,即均在6 h时有明显减少; p38 ^+/+细胞受NaAsO₂刺激后SETD4蛋白从胞质向胞核移位,而 p38 ^-/-细胞SETD4的移位现象不明显。结论: SETD4蛋白在不同种属及组织来源的细胞中均有表达,表现为全细胞分布,以胞质为主;NaAsO₂刺激可影响细胞SETD4蛋白的表达水平及定位,NaAsO₂诱导的SETD4移位改变有赖于p38 MAPK介导的信号通路。     

HIF-1α基因沉默对大鼠肝癌CBRH-7919细胞p27和Ki67表达的影响

目的: 探讨沉默缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)基因对大鼠肝癌CBRH-7919细胞在缺氧状态下p27和Ki67表达的影响。方法: 选用大鼠肝癌细胞株CBRH-7919作为研究对象,利用氯化钻(CoCl₂)模拟缺氧状态。构建HIF-1α siRNA特异性载体,利用小分子RNA干扰技术沉默肝癌细胞HIF-1α基因。应用real-time RT-PCR和Western blotting方法分别检测肝癌细胞HIF-1α mRNA和蛋白的表达变化,用Western blotting方法检测肝癌细胞 p27和Ki67 蛋白的表达变化。应用流式细胞术检测细胞周期的变化。结果: 在缺氧条件下,肝癌细胞HIF-1α mRNA及蛋白表达显著增多(P<0.05)。HIF-1α基因被沉默后,HIF-1α mRNA和蛋白表达以及Ki67蛋白表达量明显减少(P<0.05),p27蛋白表达量显著增多(P<0.05)。转染组G₀/G₁期的肝癌细胞数量明显多于对照组,S期细胞显著减少(P<0.05)。 结论: 缺氧可以诱导肝癌细胞HIF-1α的表达;特异性siRNA沉默HIF-1α,可能通过促进p27的表达和抑制Ki67的表达,在一定程度上抑制了肝癌细胞的增殖。     

上调SMYD3对人胆管癌FRH0201细胞中DNMT3B表达及细胞增殖的影响

目的: 研究人胆管癌细胞株FRH0201中SET和MYND结构域含有蛋白3(SET and MYND domain-containing protein 3,SMYD3)过度表达对DNA甲基化转移酶3B(DNA methyltransferase 3B,DNMT3B)表达及细胞增殖能力的影响。方法: 瞬时转染SMYD3真核表达质粒后, RT-PCR检测细胞中DNMT3B mRNA水平的变化;Western blotting检测细胞中DNMT3B蛋白水平的变化;CCK-8检测细胞增殖能力的改变;流式细胞术检测细胞周期的改变。结果: 以未处理组为对照,胆管癌细胞FRH0201在转染pEGFP-C3-SMYD3质粒后,DNMT3B蛋白及mRNA表达均显著上升(P<0.01);细胞的增殖能力显著提高、细胞增殖速度加快(P<0.05);进入G₂/M期的细胞明显增多(P<0.05)。结论: 过度表达SMYD3,可引起细胞中DNMT3B的表达上调并增强细胞增殖能力。     

槲皮素对毒胡萝卜素诱导的巨噬细胞内质网应激凋亡途径的抑制作用及机制

目的: 研究槲皮素(quercetin, Que)对毒胡萝卜素(thapsigargin, TG)诱导的巨噬细胞RAW264.7内质网应激凋亡途径的抑制作用及机制。方法: 1 μmol/L TG作用RAW264.7细胞24 h诱导内质网应激,不同浓度Que(80、120或160 μmol/L)与TG共同作用后,MTT法检测细胞存活率,流式细胞术检测凋亡率及[Ca²⁺]_i,激光共聚焦显微镜观察细胞形态变化,Western blotting法检测糖调节蛋白78 (glucose-regulated protein 78,GRP78)及C/EBP同源蛋白(C/EBP homologous protein, CHOP)的表达;Western blotting法检测Que(160 μmol/L)和(或)磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)抑制剂LY294002(15 nmol/L)与TG共同作用时GRP78和CHOP的表达。结果: Que能够抑制TG诱导的RAW264.7细胞内质网应激损伤,与TG组相比,细胞存活率升高(P<0.05),凋亡率降低(P<0.05),[Ca²⁺]_i降低(P<0.05),GRP78及CHOP表达减少(P<0.05);LY294002单独作用可抑制TG诱导的GRP78及CHOP表达上调(P<0.05),但与Que联合应用与二者单独使用时抑制作用无显著差异。结论: Que可以抑制TG诱导的RAW264.7细胞内质网应激凋亡途径,该作用可能与其抑制PI3K信号通路从而降低CHOP蛋白的表达有关。     

光敏化促进姜黄素诱导人胃癌MGC-803细胞凋亡

目的: 探讨光敏化促进姜黄素诱导人胃癌MGC-803细胞凋亡及其机制。方法: 用MTT法检测光敏化姜黄素对胃癌MGC-803细胞株的增殖抑制率,Hoechst 33258荧光染色观察细胞核形态的变化,流式细胞术检测细胞的凋亡率、线粒体膜电位、细胞内活性氧和Ca²⁺;比色法检测caspase-3、8和9酶活性;Western blotting分析细胞色素C、Bcl-2、Bax和热休克蛋白70(HSP70)水平。结果: 单纯姜黄素(5.0μmol/L)对MGC-803细胞增殖抑制率为(29.74±2.30)%,在光学显微镜下可见部分凋亡细胞,凋亡率为(12.54±1.75)%。而光敏化姜黄素组细胞增殖抑制率则为(44.93±3.61)%,在光学显微镜下能见明显细胞核形态改变,染色质凝集,凋亡小体形成,凋亡率为(26.58±2.67)%,细胞周期主要阻滞于G₀/G₁期。光敏化姜黄素显著降低线粒体膜电位,显著增加细胞色素C、细胞内活性氧和Ca²⁺以及caspase-3、8和9酶活性,与单纯姜黄素组比较,差异显著(P<0.01)。Western blotting结果显示光敏化姜黄素同时显著抑制Bcl-2和HSP70蛋白表达水平。结论: 光敏化姜黄素通过Bcl-2和线粒体途径增强其诱导胃癌MGC-803细胞凋亡的作用。     

人肺腺癌细胞系与支气管上皮细胞系差异蛋白质组学研究

目的:通过对肺腺癌细胞系A549细胞和正常上皮细胞系HBE蛋白组成差异的分析,寻找与肺癌发生、发展相关的差异蛋白,为进一步阐明肺癌发病机制提供更多有价值的信息;为诊断或者预后提供候选分子标记物。方法:运用固相pH梯度双向电泳和基质辅助激光解吸电离飞行时间串联质谱(MALDI/TOF/TOF/MS)分析技术筛选出A549细胞和支气管上皮细胞系HBE间表达水平显著差异的蛋白,并且利用免疫印迹方法对筛选的标记物蛋白进行验证。结果:筛选出21个差异蛋白,涉及到细胞的代谢、蛋白质修饰、细胞运动,通道蛋白和信号转导调控等方面。鉴定并且验证在A549细胞中显著高表达热休克蛋白27(HSPB1)。结论:肺腺癌细胞系与正常支气管上皮细胞系蛋白质表达存在显著差异,其中肺腺癌中高表达HSPB1可能在肺腺癌癌变过程中发挥重要作用,本结果为进一步寻找肺腺癌癌变蛋白标志物奠定了基础。     

Differential proteomic analysis of nuclear matrix in muscle-invasive bladder cancer: potential to improve diagnosis and prognosis.

INTRODUCTION: Although several molecular markers for bladder cancer have been identified, at present little information on prognostic biomarkers is available in the literature. Prognostication of this tumor is largely based on clinicopathological characteristics. Our aim was to identify nuclear matrix (NM) proteins that might serve to better characterize the phenotype of the invasive bladder cancer and to investigate their diagnostic and prognostic roles. METHODS: NM proteins expressed in normal (n=3) or non-tumoral (n=9) tissue specimens and muscle-invasive bladder cancer (n=21) specimens were analyzed by two dimensional (2D) gel electrophoresis. PDQuest image analysis software was used to generate a comparative NM proteome analysis. Selected spots were characterized by liquid chromatography coupled to tandem mass spectrometry and Western blot. RESULTS: We detected over 800 protein spots in each 2D map and 43 spots were identified. 30 proteins were differentially expressed by bladder tumor cells; among these, 19 proteins were detected in bladder tumoral tissues but not in normal and non-tumoral tissues and seven proteins correlated with tumor stage. One protein (p54nrb) was strongly correlated with vascular invasions and appeared to be also significantly (P<0.0001) associated with a decreased probability of survival. CONCLUSION: Important alterations in NM proteins occur in muscle-invasive bladder cancer. The differentially expressed proteins include biomarkers potentially useful for disease diagnosis, progression and prognosis. Our findings beyond improving the understanding of the biology of bladder cancer, could help to stratify patients into different prognostic subgroups and to select those who might be better candidate to multimodal therapeutic approaches.     

英夫利西单抗治疗克罗恩病黏膜愈合的血清差异蛋白质表达研究

目的:寻找与英夫利西单抗(infliximab,IFX)治疗克罗恩病(Crohn’s disease,CD)黏膜愈合(mucosal healing,MH)相关的血清蛋白质生物标志物。方法:采集7例经IFX治疗获得MH的CD患者治疗前(0周,A组)和治疗后(14周,B组)的血清,以及7例未获得MH的CD患者(0周为C组,14周为D组)的血清。采用荧光标记双向差异凝胶电泳的方法,比较A组与B组之间、C组与D组之间、A组与C组之间以及B组与D组之间的蛋白质组学差异,并对差异表达的蛋白质斑点进行基质辅助激光解吸/电离飞行时间串联质谱初步鉴定和生物信息学分析。结果:(1)A组与B组、C组与D组、A组与C组以及B组与D组之间比较分别存在36、3、10和31个差异表达的蛋白质斑点,共计存在44个显著差异表达的蛋白质斑点。(2)上述各组之间的差异斑点分别初步鉴定出17、2、2和15种蛋白质,共计存在19种差异表达的蛋白质,包括载脂蛋白E、载脂蛋白A-I、补体因子H等。(3)基于STRING数据库绘制了蛋白质网状功能图。结论:IFX治疗获得MH的CD患者治疗前后的血清蛋白质表达谱存在差异,获得MH和未获得MH的患者血清表达谱亦存在差异,其中19种蛋白质有可能成为预测CD患者使用IFX获得MH的生物标志物。     

Elevated expression of complement C3 protein in chemically induced hepatotumorogenesis in Wistar rats: A correlative proteomics and histopathological study

Liver cancer remains the leading cause of cancer-related mortality worldwide. Early detection of liver cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The present study was designed to determine the differently expressed proteins at early stage in the serum of animals with liver cancer vis-à-vis controls and figure out the function of the proteins. One-dimensional electrophoresis (1D), two-dimensional electrophoresis (2DE) and liquid chromatography mass spectrometry (LC–MS/MS) were used to screen the serum proteins of liver cancer induced in animals by diethyl nitrosamine (DEN) + 2-acetyl amino fluorine (2-AAF). From optimized 2DE image and computer assisted PD Quest analysis were found to be differentially expressed spots when the serum from normal and treated animals were compared. Among these, one spot was selected whose expression level was higher in DEN + 2-AAF treated animal sera than in adjacent normal animal sera. The target spot was excised from the 2D gel of liver cancer sera and the peptide mass fingerprinting as obtained LC–MS/MS analysis after digesting the chosen protein spot. This was identified to be complement C3 protein. The changes in complement C3 expression level were validated by Western blot analysis. We reported that the changes in complement C3 concentration start at very early stage of tumorogenesis. The fully grown tumors were developed at 120 days and hepatotumorogenesis was confirmed by histopathological examination. This protein may therefore represent a powerful tool in search for candidate biomarkers for HCC.     

Comparative analysis of serum proteomes: discovery of proteins associated with osteonecrosis of the femoral head

No means exist to evaluate the activity status, turnover, and prognosis of idiopathic osteonecrosis of the femoral head (IONFH) except for X-ray evidence of segmental collapse as a very good marker for prognosis. Moreover, the only current method for diagnosis of this disease is through physical examination and diagnostic imaging results, and no serum biochemical markers exist. A comparative analysis of serum proteomes was performed to discover proteins associated with osteonecrosis of the femoral head. Two-dimensional electrophoresis (2-DE) patterns of human sera from 10 patients with IONFH and 10 normal subjects were analyzed. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 7 proteins were found. The expression levels of tissue-type plasminogen activator (t-PA), bone-carboxyglutamate protein (BGP), c-sis, and an unknown protein were downregulated in the sera of patients with IONFH, whereas the other 3 proteins, including plasminogen activator inhibitor type 1 (PAI-1), crosslaps, and anti-p53 antibody, were upregulated. To examine their applicability as diagnostic markers, levels of the 6 identified proteins in serum were validated from patients with IONFH, osteoarthritis (OA), rheumatoid arthritis (RA), and fracture using the enzyme-linked immunosorbent assay (ELISA) method. It was found that only serum levels of t-PA, PAI-1, crosslaps, and anti-p53 antibody in patients with IONFH were always significantly different from those in patients with OA, RA, and fracture. These results suggest that serum levels of t-PA, PAI-1, crosslaps, and anti-p53 antibody could be used as noninvasive diagnostic biomarkers for IONFH.     

CoREST基因在骨关节炎软骨组织中的表达、分布和意义

目的: 观察RE-1元件辅助沉默因子(CoREST)基因在正常和骨关节炎(OA)膝关节软骨组织中的表达和分布,分析CoREST基因表达下调对OA的病理意义。方法: 取材膝关节正常(n=10)和OA(n=12)软骨组织,提取软骨细胞并包埋制备切片,应用real-time PCR技术对比CoREST基因在正常和OA软骨细胞中表达水平的总体差异,应用原位杂交技术观察CoREST基因在正常/OA软骨表、中、深各层组织中的表达和分布的特点。结果: CoREST基因在OA软骨细胞中的表达水平显著降低64.6%(P<0.01)。CoREST基因广泛表达于正常软骨组织的表层、中层和深层,以中层表达最为显著,其次为深层和表层。在OA软骨组织中,CoREST基因在浅层细胞簇部分细胞中表达水平升高,在深层潮线状细胞柱中表达水平普遍降低。结论: CoREST基因在正常和OA软骨组织细胞中的差异表达和分布与文献报道的X型胶原基因的组织学表达一致。结合前期研究成果,我们认为CoREST的生物功能可能与OA软骨细胞的终末分化相关。     

应用TMT结合质谱技术筛选高原红细胞增多症患者血浆差异表达蛋白

目的:应用串联质谱标签(TMT)结合质谱技术比较高原红细胞增多症(HAPC)患者和健康对照者血浆中的差异表达蛋白。方法:收集HAPC患者血浆4例,与之相匹配的健康对照组血浆5例,组内等量混合后利用多重免疫亲和层析柱去除14种高丰度蛋白,TMT试剂标记后样本进行液相色谱分离,采用HPLC-MS/MS质谱鉴定及相对定量;获取HAPC患者和健康对照血浆各20例,ELISA方法验证部分筛选的差异蛋白。结果:共鉴定和定量了1 094种蛋白,差异表达的蛋白有249种,其中HAPC组较健康对照组表达量≥1.5倍的上调蛋白质有162种,表达量≤67%的下调蛋白质有87种;C反应蛋白和血清淀粉样蛋白A在HAPC组中较健康对照组表达量均上调(P0.05)。结论:筛选出多种与炎症相关的差异表达蛋白,为深入研究HAPC发生机制奠定了良好基础。     

Proteomic analysis of synovial fluid as an analytical tool to detect candidate biomarkers for knee osteoarthritis

We conducted research to detect the proteomic profiles in synovial fluid (SF) from knee osteoarthritis (OA) patients to better understand the pathogenesis and aetiology of OA. Our long-term goal is to identify reliable candidate biomarkers for OA in SF. The SF proteins obtained from 10 knee OA patients and 10 non-OA patients (9 of whom were patients with a meniscus injury in the knee; 1 had a discoid meniscus in the knee, and all exhibited intact articular cartilage) were separated by two-dimensional electrophoresis (2-DE). The repeatability of the obtained protein spots regarding their intensity was tested via triplicate 2-DE of selected samples. The observed protein expression patterns were subjected to statistical analysis, and differentially expressed protein spots were identified via matrix-assisted laser desorption/ionisation-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Our analyses showed low intrasample variability and clear intersample variation. Among the protein spots observed on the gels, there were 29 significant differences, of which 22 corresponded to upregulation and 7 to downregulation in the OA group. One of the upregulated protein spots was confirmed to be haptoglobin by mass spectrometry, and the levels of haptoglobin in SF are positively correlated with the severity of OA (r = 0.89, P < 0.001). This study showed that 2-DE could be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA. Spots of interest identified by mass spectrometry, such as haptoglobin, may be associated with OA severity.     

Serum proteome analysis for profiling protein markers associated with carcinogenesis and lymph node metastasis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC), one of the most common cancers in population with Chinese or Asian progeny, poses a serious health problem for southern China. It is unfortunate that most NPC victims have had lymph node metastasis (LNM) when first diagnosed. We believe that the 2D based serum proteome analysis can be useful in discovering new biomarkers that may aid in the diagnosis and therapy of NPC patients. To filter the tumor specific antigen markers of NPC, sera from 42 healthy volunteers, 27 non-LNM NPC patients and 37 LNM NPC patients were selected for screening study using 2D combined with MS. Pretreatment strategy, including sonication, albumin and immunoglobulin G (IgG) depletion, was adopted for screening differentially expressed proteins of low abundance in serum. By 2D image analysis and MALDI-TOF-MS identification, twenty-three protein spots were differentially expressed. Three of them were further validated in the sera using enzyme-linked immunosorbent assay (ELISA). Our research demonstrates that HSP70, sICAM-1 and SAA, confirmed with ELISA at sera and immunohistochemistry, are potential NPC metastasis-specific serum biomarkers which may be of great underlying significance in clinical detection and management of NPC.     

Proteomic analysis of mitral valve in Lewis rat with acute rheumatic heart disease

Rheumatic heart disease (RHD) makes a heavy burden in human lives and economy. The proteomic analysis of acute rheumatic heart disease (ARHD) can provide precious data to study RHD at the early stages, but no one has looked into. So based on our early research we applied the method of continuous GAS stimulation on Lewis rats to duplicate the animal model of ARHD. And the mitral valves of rats in control group (n=10) and ARHD group (n=10) were selected for proteomic analysis of ARHD with the iTRAQ labeling based 2D LC-ESI-MS/MS quantitative technology. We identified 3931 proteins in valve tissue out of which we obtained 395 differentially expressed proteins containing 176 up-regulated proteins and 119 down-regulated proteins. Changes in levels of GAPDH (6.793 times higher than the control group) and CD9 (2.63 times higher than the control group) were confirmed by Western blot or immunohistochemistry. The differentially expressed proteins such as GAPDH, CD9, myosin, collagen and RAC1 may be potential biomarkers for ARHD. Moreover, the mitral valve protein profile shed light on further understanding and investigating ARHD.