类风湿关节炎患者外周血单个核细胞蛋白质组学研究

目的 运用蛋白质组学的方法,比较正常人及早期活动性类风湿关节炎(RA)患者外周血单个核细胞(PBMCs)蛋白质的差异表达,寻找RA疾病相关致病蛋白质.方法 选取9例早期活动期RA患者以及9名健康成年志愿者,用淋巴细胞分离液分离PBMCs,抽提PBMCs中的蛋白,采用同相pH梯度(IPG)双向凝胶电泳(2-DE)分离正常人及RA患者PBMCs总蛋白质.凝胶经考马斯亮蓝染色显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,对差异蛋白质点用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)进行鉴定,运用反转录-聚合酶链反应(RT-PCR)方法验证部分差异蛋白质.结果 获得RA患者及正常人PBMCs蛋白质双向凝胶电泳图谱,平均蛋白质点数分别为556和579,匹配率分别为89.4%和 88.5%,通过比较分析,差异表达蛋白质点数为23,选取18个点进行质谱鉴定,成功鉴定14个蛋白质,其中a-肌动蛋白、纤维蛋白素原a-链、载脂蛋白A-I(ApoA-I)等9个蛋白质点在RA中表达上调,硫氧还蛋白-2、谷胱甘肽S-转移酶等6个蛋白质点在RA中表达下调,这些差异蛋白质的功能涉及物质代谢、抗氧化、信号传导、能量产生及细胞骨架.并用RT-PCR方法验证差异蛋白质ApoA-I.其结果与上述蛋白质差异表达结果相符.结论 在RA患者PBMCs中存在着差异表达蛋白质,这些差异蛋白质可能是RA发病的内在因素.其RT-PCR结果与蛋白质差异表达相符,证明蛋白质组研究的可靠性.     

超短肠大鼠结肠代偿相关蛋白筛选与鉴定

目的　筛选并鉴定与结肠代偿相关的蛋白质。方法　SD大鼠20只,随机分为两组,每组10只。超短肠组：制作切除90%小肠的超短肠大鼠;对照组：横断小肠后再吻合。术后喂养21 d,提取结肠黏膜蛋白质,通过双向电泳,建立各组蛋白质双向电泳图谱,对异蛋白斑点进行MALDI-TOF/TOF-MS鉴定,通过查找数据库分析差异蛋白功能。结果　建立了超短肠组、对照组结肠黏膜蛋白的双向电泳图谱,通过质谱技术鉴定出4个差异蛋白：前梯度同源蛋白2、丙酮酸激酶同工酶、蛋白酶激活剂复合物亚型1、胰甘油三酯酯酶,功能涉及糖和脂肪代谢、细胞增殖等。结论　鉴定出的蛋白质可能通过加强物质代谢、促进细胞增殖等途径参与结肠代偿。     

胸膜斑阳性接尘工人血清效应蛋白的研究

目的 监测胸膜斑阳性接尘工人血清效应蛋白质,探讨胸膜斑发病机制及其与石棉肺的关系.方法 将研究对象分为胸膜斑组、阴性对照组和正常对照组.各组人群静脉血应用双向电泳和质谱进行分离和鉴定,以灰度值差异1.5倍为基准获得差异表达蛋白质.结果 胸膜斑组与阴性对照组和正常对照组比较,差异点分别有34和16个;阴性对照组与正常对照组比较,差异点有32个.效应蛋白质如补体因子、α1-抗胰蛋白酶等参与机体免疫、抗肿瘤等过程.部分效应蛋白质与石棉肺血清效应蛋白一致.结论 长期接触石棉导致血清中某些蛋白质表达量改变,进而影响其生物学功能.胸膜斑和石棉肺有相同的靶效应蛋白,提示胸膜斑工人更易患石棉肺.     

The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing

Aims

In this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level.

Methods

Spectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)₄, and (AC)₁₀ was performed in order to assess the intra-specific variability of Trichophyton rubrum strains.

Results

The identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%.

Conclusions

MALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubrum.     

Relationship between E - cadherin and diabetic nephropathy

Objective To identify novel biomarker for diabetic nephropathy (DN) by urinary proteomic methods, and to detect the expression of E-cadherin in urine and renal tissue of patients with DN. Methods Urine samples were collected from 12 cases of type 1 diabetic nephropathy patients (T1DN), 12 cases of type 2 diabetic nephropathy patients (T2DN), 12 cases of nephritic syndrome patients (NS), and 12 cases of healthy Controls. Comparative proteomic approach of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were employed to identify DN-related biomarker in urine samples. The differential expression of the identified biomarker in urine samples and renal biopsy specimens were detected by Western blotting and immunohistochemistry method. Results E-cadherin was identified by 2DE/MS, which was significantly up-regulated in T1DN and T2DN groups (all P＜ 0.01). Western blotting confirmed the expression of E-cadherin was significantly higher in T1DN and T2DN groups than in NS and Control groups (all P＜0.01). Immunohistochemical stain showed E-cadherin was mainly expressed in the membrane and cytoplasm of renal tubular epithelial cell, and its expression was markedly decreased in DN kidneys compared with healthy Controls (P＜0.05). Conclusions E-cadherin is identified as a novel DN-related biomarker, which is specifically increased in urine of DN patients.     

The molecular mechanism for effects of TiN coating on NiTi alloy on endothelial cell function

The aim of this study is to systematically investigate the molecular mechanism of different effects of nickel titanium (NiTi) alloy surface and titanium nitride (TiN) coating on endothelial cell function. Release of nickel (Ni) ion from bare and TiN-coated NiTi alloys and proliferation of endothelial cells on the two materials were evaluated, and then influence of the two materials on cellular protein expression profiles was investigated by proteomic technology. Subsequently, proteomic data were analyzed with bioinformatics analyses and further validated using a series of biological experiments. Results showed that although the two materials did not affect cell proliferation, the Ni ions released from bare NiTi alloy generated inhibition on pathways associated with actin cytoskeleton, focal adhesion, energy metabolism, inflammation, and amino acid metabolism. In comparison, TiN coating not only effectively prevented release of Ni ions from NiTi alloy, but also promoted actin cytoskeleton and focal adhesion formation, increased energy metabolism, enhanced regulation of inflammation, and promoted amino acid metabolism. Furthermore, the two processes, “the initial mediation of adsorbed serum protein layer to endothelial cell adhesion and growth on the two materials” from our previous study, and “the following action of the two materials on cellular protein expression profile”, were linked up and comprehensively analyzed. It was found that in stage of cell adhesion (within 4 h), release of Ni ions from bare NiTi alloy was very low, and the activation of adsorbed proteins to cell adhesion and growth related biological pathways (such as regulation of actin cytoskeleton, and focal adhesion pathways) was almost as same as TiN-coated NiTi alloy. This indicated that the released Ni ions did not affect the mediation of adsorbed proteins to endothelial cell adhesion. However, in stage of cell growth and proliferation, the release of Ni ions from bare NiTi alloy increased with time and reached a higher level, which inhibited endothelial cell function at molecular level, whereas TiN coating improved endothelial cell function.     

Proteomic Analysis of Saliva from Patients with Oral Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder and is the major long-term complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The oral mucosa, including the salivary glands, is affected in the majority of patients with cGVHD; however, at present there is only a limited understanding of disease pathobiology. In this study, we performed a quantitative proteomic analysis of saliva pooled from patients with and without oral cGVHD—cGVHD(+) and cGVHD(−), respectively—using isobaric tags for relative and absolute quantification labeling, followed by tandem mass spectrometry. Among 249 salivary proteins identified by tandem mass spectrometry, 82 exhibited altered expression in the oral cGVHD(+) group compared with the cGVHD(−) group. Many of the identified proteins function in innate or acquired immunity, or are associated with tissue maintenance functions, such as proteolysis or the cytoskeleton. Using ELISA immunoassays, we further confirmed that 2 of these proteins, IL-1 receptor antagonist and cystatin B, showed decreased expression in patients with active oral cGVHD (P < .003). Receiver operating curve characteristic analysis revealed that these 2 markers were able to distinguish oral cGVHD with a sensitivity of 85% and specificity of 60%, and showed slightly better discrimination in newly diagnosed patients evaluated within 12 months of allo-HSCT (sensitivity, 92%; specificity 73%). In addition to identifying novel potential salivary cGVHD biomarkers, our study demonstrates that there is coordinated regulation of protein families involved in inflammation, antimicrobial defense, and tissue protection in oral cGVHD that also may reflect changes in salivary gland function and damage to the oral mucosa.     

大鼠脑组织蛋白质双向电泳技术的建立

目的：建立大鼠脑组织蛋白质双向电泳技术。方法：利用不同体积的裂解液提取大鼠脑组织中的蛋白质,并通过不同的蛋白质上样量（1mg,2mg,3mg）,进行双向电泳,考马斯亮蓝染色,图谱分析。结果：在等电点3～10,分子量6．5～200ku范围内分离得到蛋白质斑点为,1mg蛋白质上样量为546个蛋白质斑点,2mg为780个,3mg为805个斑点。2mg蛋白质上样量的双向电泳图谱更清晰,分离更好。结论：成功建立了大鼠脑组织蛋白质的双向电泳技术。     

Alteration of protein profile in cerebral cortex of rats exposed to bisphenol a: a proteomics study

Bisphenol A (BPA) is one of the most widely used chemicals in plastic industry, which enters the human body through occupational and food contact. We studied the protein changes in rat cerebral cortex to evaluate the neurotoxicity of BPA. Twenty-four adult male rats were randomly selected and divided into four groups and each group respectively received 0, 0.5, 5 and 50 mg/kg of BPA for 4 weeks orally. To determine the oxidative status, reduced glutathione content and the level of malondialdehyde were measured in brain cortical tissue. The proteins of each sample extracted and separated on a two-dimensional acrylamide gel electrophoresis. From the obtained protein map, the 10 most altered protein spots were used for mass spectroscopy analysis. The lipid peroxidation in both doses of 0.5 and 5 mg/kg was significantly higher than the control group, but the glutathione content had no significant difference between the groups. Based on the results of the MS data analysis by the MASCOT database search engine, 10 proteins with altered intensity were identified as pyruvate kinase, alpha-enolase, aconitate hydratase, creatine kinase B-type, phosphatidylethanolamine-binding protein 1, 14-3-3 protein eta, guanine nucleotide-binding protein subunit beta-1, dihydropyrimidinase-related protein 2, glutamine synthetase and the neurofilament light polypeptide. There are several reports suggesting that the increase or decrease in the level and activity of these 10 proteins, similar to those observed in this study, is related to some neurological and psychosocial disorders including neurodegenerative diseases, schizophrenia, depression, epilepsy and some brain tumors.