基于iTRAQ技术筛选肝细胞癌微血管侵犯的生物标记物

目的 利用核素标记的相对和绝对定量(iTRAQ)技术比较肝内无血管侵犯和微血管侵犯的肝细胞癌(HCC)患者的血清,筛选并鉴定其差异表达的蛋白质,以作为HCC微血管侵犯的生物标记物.方法 收集无血管侵犯和微血管侵犯的HCC患者血清各15例,组内等量混合后利用免疫亲和柱去除14种高丰度蛋白,iTRAQ试剂标记后的样本利用二维色谱分离,经串级质谱鉴定及相对定量.结果 鉴定得到75种蛋白质,其中差异表达的蛋白有20种(上调或下调20%),微血管侵犯组较无血管侵犯组上调的蛋白有12种,下调的有8种.结论 鉴定出多种与肝癌微血管侵犯相关的生物标记蛋白,为肝细胞癌转移和复发的预测和术后的干预治疗提供了实验依据.     

一种优化改良的胆汁蛋白质组学研究方法

目的:探索一种可靠、方便、高通量的胆汁蛋白质组学研究的方法。方法:抽取3例胆囊结石和3例胆囊癌患者的胆囊胆汁进行蛋白质提取纯化和定量,然后利用高通量的蛋白质组学研究方法同位素标记相对和绝对定量(iTRAQ)进行蛋白质的鉴定以及定量,并进行生物信息学分析。结果:经浓度和完整性检测,提取的胆汁蛋白质样品的浓度和完整性均达到了iTRAQ实验的要求。经过鉴定,共检测出1 323种蛋白质,比传统方法检测出的蛋白质数量显著提高。与结石患者胆汁比较,肿瘤患者胆汁有173种蛋白质明显上调,有345种蛋白质明显下调(变化倍数1.5,P0.05)。结论:建立了一种可靠的胆汁蛋白质分离、纯化、鉴定、分析的方法,鉴定出的蛋白质数量比传统方法明显增多,更有潜力发现与疾病相关的蛋白质分子,为日后胆汁以及其他体液的蛋白质组学研究奠定了基础。     

原发性肝癌组织分泌蛋白同位素标记相对和绝对定量技术研究

肝细胞癌(HCC)的早诊早治是提高生存率的关键,分泌蛋白在筛选肿瘤标志物方面有很大的价值。本研究利用同位素标记相对和绝对定量技术(iTRAQ)与液相色谱-质谱联用技术(LC-MS/MS)结合的定量蛋白组学研究HCC的分子特征。     

业界专家谈科学构建生物样本资料库

生物样本库是一种集中储存各种人类生物样本如血液、组织等,用于疾病临床治疗和生命科学研究的生物应用系统,在心血管疾病、恶性肿瘤等重大疾病的新药研制、探索诊疗方法、制定新的诊断标准等领域拥有极其重要的战略意义.随着转化医学的迅速发展,高质量的生物研究样本对于探索新的诊疗方法,确定新的诊断指标以及新药的研发,日益凸显其重要意义.目前,我国大规模临床研究和流行病学研究数量庞大,研究样本资源丰富,但符合国际标准、高质量、大容量的生物样本库,却明显匮乏,这在一定程度上制约了我国生物医学研究的长期、可持续发展.     

基于iTRAQ技术研究桃核承气汤对UUO大鼠肾组织蛋白质组学的影响

目的:应用同位素标记相对和绝对定量(iTRAQ)技术研究桃核承气汤对单侧输尿管梗阻(UUO)大鼠肾组织蛋白质组学的影响,探讨桃核承气汤延缓肾间质纤维化(RIF)的作用机制。方法:30只SPF级wistar雄性大鼠,随机分为正常组、UUO组及桃核承气汤组,各10只,除正常组外均采用UUO法造模。术后第1天开始,桃核承气汤组给予灌服5 ml桃核承气汤水煎液;UUO组给予灌服5 ml蒸馏水,每日灌服1次。给药后14 d取肾组织,镜下观察肾组织纤维化程度,并基于iTRAQ标记技术进行蛋白质组学检测,以模型组均值/正常组均值大于1.2或小于0.83为标准,T检验P值小于0.05为差异蛋白,并对差异蛋白进行生物信息学分析。结果:通过蛋白质组学检测共鉴定出343个差异蛋白,其中上调的蛋白251个,下调的蛋白92个,蛋白之间相互作用构成网络,涉及多个生物学过程。结论:应用iTRAQ技术筛选出桃核承气汤治疗后大鼠肾组织中差异蛋白,深入研究桃核承气汤延缓RIF的分子机制。     

应用定量蛋白质组学技术对狼疮肾炎组织蛋白的鉴定和定量分析

目的应用定量蛋白质组学技术对狼疮肾炎（LN）V型肾组织中的“全组”蛋白进行鉴定和定量分析，获得LNV型的蛋白质组差异表达图谱。方法对LNV型肾组织及对照组进行相对和绝对定量的同位素（isobaric tags for relative and absolute quantitation, iTRAQ）标记和ESI-OJTOF质谱仪鉴定。结果共鉴定490个蛋白质，其中差异倍数1．0以上的蛋白数113个，差异倍数1．2以上的蛋白数58个，差异倍数1．5以上的蛋白数18个，表达上调和下调的蛋白分别为10个和8个。结论定量蛋白质组学技术可有效地用于组织蛋白鉴定和相对定量；采用该技术获得LNV型肾组织蛋白质组差异表达图谱；深入研究这些蛋白的分子机制将有助于进一步阐明LNV型的发病机制及发现的潜在标志物，为LN的诊断和治疗提供新途径。     

颅面多层CT三维影像重建的研究

目的建立颅面计算机辅助三维重建技术.方法对男女各20例正常人颅面形态结构做多层CT三维影像立体测量.结果所得重建图像逼真,可任意剖面和多视觉变换,显示颅面各结构的空间位置关系和定量测量分析.结论计算机辅助三维重建技术为颅面结构特征的研究提供了新的手段,并构建了一种新的形态数据资料的储存方式.     

颅面多层CT三维影像重建的研究

目的 建立颅面计算机辅助三维重建技术。方法 对男女各20例正常人颅面形态结构做多层CT三维影像立体测量。结果 所得重建图像逼真，可任意剖面和多视觉变换，显示颅面各结构的空间位置关系和定量测量分析。结论 计算机辅助三维重建技术为颅面结构特征的研究提供了新的手段，并构建了一种新的形态数据资料的储存方式。     

基于iTRAQ技术探讨水蛭干预IgA肾病大鼠模型蛋白质组学研究

目的运用iTRAQ结合质谱技术检测IgA肾病(IgAN)大鼠模型尿液蛋白指纹图谱,筛选差异表达蛋白,研究水蛭治疗血瘀证IgAN的作用机制。方法选用6周龄的Wistar雄性大鼠140只,运用脂多糖牛血清白蛋白+脂多糖+四氯化碳方法建立实验性IgAN模型,造模成功后,随机分为模型对照组(不给予干预)、生理盐水对照组(给予生理盐水2 mL/100 g灌胃)、水蛭高剂量治疗组(给予泼泥松5.5 mg·kg~(-1)·d~(-1)+水蛭1.2 g·kg~(-1)·d~(-1)灌胃。每日一次)、水蛭中剂量治疗组(给予泼泥松5.5 mg·kg~(-1)·d~(-1)+水蛭0.6 g·kg~(-1)·d~(-1)灌胃。每日一次)、水蛭低剂量治疗组(给予泼泥松5.5 mg·kg~(-1)·d~(-1)+水蛭0.3 g·kg~(-1)·d~(-1)灌胃。每日一次)、泼尼松治疗对照组(给予泼尼松5.5 mg·kg~(-1)·d~(-1)),另设立空白对照组,每组20只。收集各组大鼠尿液样本,经过SDS-PAGE电泳后,进行iTRAQ标记,采用离线二维液相色谱分离与点靶以及质谱分析,运用数据库检索对各组样本进行相对定量分析,并利用生物信息学软件进行信息学分析。结果以P值0.05且蛋白丰度差异0.83或蛋白丰度差异1.2为差异蛋白筛选标准,共筛选出的16个差异蛋白,其中模型对照组和空白对照组相比上调、水蛭治疗后下调的蛋白有6个,模型对照组和空白对照组相比下调、水蛭治疗后上调的蛋白有10个。采用KEGG富集分析筛选出和IgAN发生、发展密切相关的有10条分子信号通路,主要通路为补体和凝血级联途径。结论应用iTRAQ技术筛选出水蛭治疗后IgAN大鼠模型尿液标本中差异表达蛋白及与IgAN发生、发展密切相关的信号通路,为深入研究IgAN血瘀证的发生机制及水蛭治疗IgAN的作用机制奠定了基础。     

基于iTRAQ技术的BMP-2诱导肌源C2C12细胞向成骨细胞分化过程中的蛋白质组学研究

 目的 应用iTRAQ技术观察BMP-2诱导肌源C2C12细胞向成骨细胞分化过程中蛋白质表达组的变化。方法 将肌源C2C12细胞接种于BMP-2诱导分化体系中进行分化诱导,提取第7天的分化蛋白以iTRAQ试剂标记后进行质谱检测,分析差异表达的蛋白质,并进行生物信息学分析。结果 iTRAQ 试剂标记的BMP-2诱导肌源C2C12分化细胞蛋白质表达谱分析筛选出明显差异表达蛋白质点为23个,表达上调的蛋白质点8个,下调的蛋白质点15个。通过趋势分类发现上述差异蛋白在C2C12细胞成骨分化的各时期（第1至7天）均存在蛋白的差异表达。其中部分上调蛋白在分化早期表达水平升高、部分上调蛋白在分化晚期表达水平升高;同样,部分下调蛋白在分化早期表达水平下降,部分下调蛋白在分化晚期表达水平下降。初步鉴定SERCA3、细胞色素b5、S100A4、ATPase inhibitor、ATPIF1等5个蛋白表达出现动态变化,证实上述蛋白可能与诱导成骨分化机制有关。结论 本研究差异蛋白表达趋势的结果显示全程监测C2C12细胞成骨分化的必要性,提示iTRAQ技术是研究细胞分子蛋白改变的有效的蛋白质组学方法,SERCA3、细胞色素b5、S100A4、ATPase inhibitor、ATPIF1等5个蛋白可作为诱导成骨分化机制研究的候选靶标。     

Identification of serum biomarkers in brain-injured adults: potential for predicting elevated intracranial pressure

Brain injury biomarkers may have clinical utility in stratifying injury severity level, predicting adverse secondary events or outcomes, and monitoring the effectiveness of therapeutic interventions. As a biomarker source, serum offers several advantages over cerebrospinal fluid (CSF), including ease of accessibility and reduced risk to the patient. We screened pooled serum samples obtained from 11 severely injured traumatic brain injury (TBI) patients (Glasgow Coma Scale [GCS] 25 mm Hg). Our results support the use of serum as a source for discovery of TBI biomarkers, and indicate that serum biomarkers may have utility for predicting secondary pathologies (e.g., elevated ICP) associated with TBI.     

Chd4 and associated proteins function as corepressors of Sox9 expression during BMP‐2–induced chondrogenesis

Mouse embryonic fibroblasts (MEFs) differentiate into fully functional chondrocytes in response to bone morphogenetic protein‐2 (BMP‐2). However, the comprehensive proteomic aspect of BMP‐2–induced chondrogenesis remains unknown. We took advantage of quantitative proteomic analysis based on isobaric tag for relative and absolute quantitation (iTRAQ) and on‐line 2D nano‐liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify proteins differentially expressed during BMP‐2–induced chondrogenic differentiation of MEFs. We found 85 downregulated proteins, and ingenuity pathways analysis (IPA) revealed a protein‐protein network with chromodomain‐helicase‐DNA‐binding protein 4 (Chd4) in the center. Chromatin immunoprecipitation (ChIP) and nuclease hypersensitivity assays showed that Chd4, interacting with Hdac1/2, cooperates with its related proteins Kap1 and Cbx1 to bind at ?207/?148 of the Sox9 promoter. We also provided evidence that let‐7a targets the 3'UTR of Chd4 to promote chondrogenesis of MEFs. Together, our findings indicate that BMP‐2 induced the upregulation of let‐7a, targeting Chd4 and positively controlling the chondrogenic differentiation of MEFs. These findings illustrate epigenetic regulation of the chondrogenic differentiation process and also expand the understanding of the involved intracellular mechanisms.     

腰椎椎间盘突出症临床评估量表的建立及前瞻性研究

目的建立腰椎椎间盘突出症临床评估量表,通过评价患者手术短期疗效对其加以验证。方法 2010年3月～2010年9月50例经手术治疗的单节段腰椎椎间盘突出症患者纳入本次前瞻性研究。根据病史和影像学等资料,制定腰椎椎间盘突出症临床评估量表。记录患者临床评估量表主观症状得分,客观体征得分,影像学和社会心理等因素得分。采用Stauffer-Coventry术后疗效评定标准作为一种评价指标,本标准术后疗效分优、良、中、差4个等级。结果本组患者均获随访,时间为12～18个月（平均15个月）。每例患者临床评估量表评分的总分均≥60分,主观症状评分均≥13分,社会心理因素评分均≥9分,客观体征评分均≥10分,影像学评分均≥17分。50例中优42例,占84%;良8例,占16%;无复发患者。结论腰椎椎间盘突出症患者临床评估量表有助于筛选适合手术的病例,提高手术疗效;但仍需大样本资料证实其准度和效度,进一步完善此临床评估量表。     

The potential role of heat shock proteins in acute spinal cord injury

Purpose

This study aims to investigate the differential expression proteins profile of spinal cord tissues after acute spinal cord injury (ASCI), provide preliminary results for further study and explore the secondary injury mechanisms underlying ASCI.

Methods

Using Allen’s frame to establish ASCI model of Sprague–Dawley rats, then a stable isotope-labelled strategy using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional (2D) liquid chromatography tandem mass spectrometry (2D LC–MS/MS) was performed to separate and identify differentially expressed proteins.

Results

A total of 220 differentially expressed proteins were identified in the spinal cord tissues of H-8 group (acute spinal cord injury after 8 h) compared with H-0 group (acute spinal cord injury after 0 h); Up to 116 proteins were up-regulated, whereas 104 proteins were down-regulated in the spinal cord tissues. Three of the differentially expressed Heat shock proteins (HSPs) namely, Hsp90ab1, Hspa4 and Hspe1 were down-regulated.

Conclusion

The differentially expressed proteins of spinal cord tissues after ASCI will provide scientific foundation for further study to explore the secondary injury mechanism of ASCI.     

基于iTRAQ技术的WB-F344细胞向胆管细胞分化过程中的蛋白质组学研究

目的 应用iTRAQ技术观察WB-F344细胞向胆管细胞分化过程中蛋白质表达组的变化.方法 将WB-F344细胞接种于丁酸钠分化体系中进行分化诱导,提取不同时间点(第0、2、4、6天)的分化蛋白以iTRAQ试剂标记后进行质谱检测并以软件分析差异表达的蛋白质.结果 iRAQ试剂标记的WB-F344分化细胞蛋白质表达谱分析...     

An in vitro method of studying functional responses of penile resistance arteries under isobaric conditions

PURPOSE: We developed an in vitro method that allows us to study the physiopharmacological responses of penile resistance arteries under isobaric conditions. MATERIALS AND METHODS: Second to third order penile resistance arteries (internal diameter 170 to 210 microm) were mounted in a pressure myograph and cannulated at each end with small glass cannulas (tip external diameter 150 to 180 microm). Internal diameter was continuously recorded and monitored under an intraluminal pressure of 60 mm Hg. RESULTS: Noradrenaline (0.1 to 0.3 microM) induced a decrease in the luminal diameter of the penile arteries, ie vasoconstriction. This effect was reversed by 1 microM acetylcholine, 1 microM prostaglandin E1 (PGE1) and 1 nM to 1 microM sildenafil citrate. Furthermore, the vasodilatation induced by sildenafil was compared by artery internal diameter values under isometric and isobaric conditions. Although the mean potency of this drug +/- SEM, expressed in pD2, was higher in 5 isometric (7.60 +/- 0.04) than in 4 isobaric (7.03 +/- 0.20) preparations (p <0.05), the slope of the curve was lower in 4 isobaric (0.49 +/- 0.02) than in 5 isometric (1.34 +/- 0.11) studies (p <0.01). CONCLUSIONS: Under isobaric conditions all vasoactive agents tested inhibited the noradrenaline induced vasoconstriction. Furthermore, the vasodilatory effect of PGE1 beyond baseline diameter could suggest an inhibitory effect of PGE1 on spontaneous myogenic tone. On the other hand, the effect of sildenafil was more potent under isometric than under isobaric conditions. However, the lower slope of the curve under isobaric conditions suggests that the pressure myograph could be a more suitable in vitro model for the study of the activity of penile resistance arteries, and so isobaric conditions correspond more closely to the in vivo situation.     

The incidence of transient neurological symptoms after spinal anaesthesia with lidocaine compared to prilocaine

The purpose of this double-blind study was to investigate the incidence of transient neurological symptoms after the use of isobaric lidocaine and isobaric prilocaine for spinal anaesthesia. Seventy patients (ASA 1-2, age between 18 and 70 years) were randomly assigned to two groups of 35 patients each, to receive either isobaric 2% lidocaine 4 ml or isobaric 2% prilocaine 4 ml intrathecally, at the L3-4 interspace. One patient in the prilocaine group could not be included because data were incomplete. On the first postoperative day, patients were evaluated for transient neurological symptoms. Pain was scored on a 10-point scale. Seven patients (20%) in the lidocaine group had transient neurological symptoms with a mean pain score of 5.3, whereas no patient in the prilocaine group had these complaints (p = 0.006). Symptoms disappeared within 4 days. Prilocaine results in a lower incidence of transient neurological symptoms than lidocaine intrathecally and therefore it is more suitable for short surgical procedures.