便秘型肠易激综合征结肠黏膜组织蛋白质组双向凝胶电泳分析

目的:分析便秘型肠易激综合征(C-IBS)患者与正常人结肠黏膜组织蛋白质表达的差异．方法:采用双向凝胶电泳(2-DE)技术和计算机辅助的图像分析方法,对C-IBS患者与正常人结肠黏膜组织蛋白质进行分离和比较分析．结果:正常对照组平均胶蛋白斑点数为308,C-IBS组平均胶蛋白斑点数为238,与正常对照组平均胶匹配点数为178,匹配率74．79％．有18个蛋白质点的表达量存在明显差异,其中有3个蛋白点表达发生明显上调,有15个蛋白点表达发生明显下调．结论:C-IBS患者与正常人结肠黏膜组织蛋白质表达存在明显差异,可能与C-IBS的发病机制有关．     

HBV相关性肝细胞癌及肝硬化患者外周血单个核细胞质谱分析

目的:建立HBV相关性肝细胞癌(HCC)及肝硬化(LC)患者外周血单个核细胞(PBMC)的双向凝胶电泳图谱,并识别鉴定其差异表达的蛋白质.方法:利用固相pH梯度双向凝胶电泳分离HBV相关性HCC(n=16)及LC患者(n=12)PBMC的总蛋白质,凝胶经蓝银显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到相应的肽质指纹图谱,然后搜索数据库鉴定部分差异蛋白质点.结果:得到了分辨率较高、重复性较好的HBV相关性HCC及LC患者PBMC双向凝胶电泳图谱,HBV相关性HCC及LC患者PBMC凝胶的平均蛋白质点数分别为1186±43及1013±41;组内的平均匹配率分别为91.6%,90.2%.对27个差异蛋白质点进行了质谱分析,鉴定出16个蛋白质,在HBV相关性HCC患者PBMC中表达明显增强的有热休克蛋白质(HSP)27、ras癌基因家族(RAB14)、肌动蛋白、α1-抗胰蛋白及RNA结合蛋白调节亚基等13个蛋白点;而PDX6、HSPA8及锰超氧化物歧化酶(MnSOD)在HCC患者PBMC中低表达.这些差异蛋白质点包括蛋白质合成与分解、分子伴侣、解毒和DAN修复相关蛋白质、三大代谢相关酶类、细胞结构相关蛋白质以及信号传导相关蛋白质六类.结论:建立了分辨率高且重复性较好的HBV相关性HCC及LC患者PBMC双向凝胶电泳图谱,并识别鉴定出了一些与HBV相关性HCC癌变相关的差异表达的蛋白质.     

维吾尔族原发性高尿酸血症血清差异蛋白质研究

采用双向凝胶电泳和基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)对维吾尔族高尿酸血症人群和对照组人群血清进行差异蛋白质研究,并用Western印迹法进行差异蛋白质验证.结果显示,差异表达蛋白质点数为11个,质谱成功鉴定出4个差异蛋白质,分别是补体C3、触珠蛋白、补体C4和载脂蛋白A1,均呈上调表达.Western印迹法验证了补体C3在2组人群中的差异与蛋白质组学结果相一致.     

胶原诱导性关节炎大鼠滑膜病变的蛋白质组学研究

目的探讨胶原诱导性关节炎(CIA)大鼠滑膜病变的蛋白质组学研究,为类风湿关节炎(RA)的发病及治疗提供新的线索。方法30只采用皮下注射牛Ⅱ型胶原与完全弗氏佐剂诱导的SD大鼠CIA诱导性关节炎(CIA)模型,应用双向凝胶电泳(2DE)技术分离正常组、CIA模型组大鼠关节滑膜的总蛋白后,经胶体考染显色、图像分析、识别差异表达的蛋白质点,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到相应的肽质量指纹图谱(PMF),然后搜索数据库鉴定部分差异蛋白质点,运用western blotting方法验证差异表达蛋白质。结果建立了正常组、CIA模型组大鼠滑膜的双向凝胶电泳图谱,平均点数为(1020±40)个蛋白质点,平均匹配率为(93．2±2．1)%；发现并鉴定了两组滑膜差异表达大于2倍的蛋白质点35个,这些与滑膜病变相关的差异表达蛋白质的功能涉及物质代谢、能量产生、物质转运、抗氧化、信号转导及细胞骨架蛋白,随即用western blotting方法验证差异蛋白质annexinⅠ、aldolase A在正常组和CIA模型组中的表达,其结果也显示了类似的表达差异。结论建立了正常组、CIA模型组大鼠关节滑膜的双向凝胶电泳图谱,鉴定了35个与CIA滑膜病变相关的差异表达的蛋白质,这些差异蛋白质可能以不同的方式参与了病变过程,为揭示CIA滑膜病变的机制及RA的发病机制提供了新的线索,而且annexinⅠ、aldolase A的验证结果与蛋白质组结果的一致性表明,AnnexinⅠ、aldolase A可能与CIA的关节滑膜病变有关。     

怀化侗族高血压人群IL-1Ra、CK-18、α-烯醇化酶蛋白质表达差异

目的分析怀化侗族高血压患者血淋巴细胞蛋白质谱,寻找高血压患者血淋巴细胞差异蛋白质。方法应用MALDI-TOF-MS技术检测130例血淋巴细胞标本(侗族高血压50例,汉族高血压40例,侗族正常人群40例)的蛋白质质谱,用MatrixScience公司的Mascot对蛋白质质谱数据进行数据库查询比对,并搜索鉴定蛋白。结果三组均获得重复性好的血淋巴细胞蛋白质双向凝胶电泳图谱。通过对其中3个差异表达蛋白点分析,并经质谱鉴定。与侗族正常人对照,侗族高血压人群α-烯醇化酶表达上调,CK-18表达差异无统计学意义(P﹥0.05),IL-1Ra表达下调;汉族高血压人群CK-18、α-烯醇化酶两个点表达上调,IL-1Ra表达下调。结论 IL-1Ra和α-烯醇化酶可作为预测高血压发生、发展程度的一个候选生物学标志物。     

三水白虎汤对类风湿关节炎滑膜成纤维细胞增殖的蛋白质组学影响

目的 探讨三水白虎汤(SSBH)对类风湿关节炎(RA)滑膜成纤维细胞(FLS)增殖的蛋白质组学影响.方法 组织块培养法收集RA患者的关节FLS,体外传代培养,取3～6代细胞,分别加入SSBH含药血清培养(SSBH组)及加入生理盐水制备的血清培养(对照组).培养72 h后提取细胞总蛋白,用双向凝胶电泳(2-DE)分离细胞总蛋白,凝胶经银染显色和图像数据分析识别差异蛋白位点;基质辅助激光解析电离飞行时间质谱对差异蛋白位点进行鉴定.结果 两组FLS蛋白2-DE图谱均出现差异蛋白质点.在差异蛋白表达量超过3倍的蛋白质点中,选取27个差异蛋白位点进行质谱分析,共鉴定出包括表达上升的IL-1受体拮抗蛋白等25种蛋白质.结论 SSBH可能通过上调RA患者FLS中IL-1受体拮抗蛋白来抑制滑膜增生,这可能是该方剂治疗RA的药理作用机制之一.     

系统性红斑狼疮患者外周血单个核细胞蛋白质组学研究

目的 分析系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)蛋白质表达谱的变化.方法 分别抽取SLE患者及健康对照外周血,分离单个核细胞,抽提总蛋白,进行双向电泳、电泳胶银染显色,用ImageMaster 2D Platinum 5.0软件对获得的蛋白图谱进行分析,寻找差异表达的蛋白质.利用基质辅助激光解析电离飞行时间质谱鉴定差异蛋白点.结果 对照组凝胶蛋白点匹配率为(71±4)%,患者组匹配率为(72±4)%.对照组凝胶共检出蛋白点(791±17)个,患者组检出(781±17)个.有11个蛋白点在SLE患者组表达上调,9个表达下调,质谱分析共鉴定5个蛋白.以前的研究显示,我们鉴定的部分蛋白在SLE的发病机制中起潜在的作用.结论 SLE患者外周血单个核细胞蛋白质表达发生了明显改变,为我们从淋巴细胞蛋白质谱变化的整体角度上阐明SLE发生的分子机制及免疫调控通路奠定了基础.     

肺结核患者外周血单个核细胞蛋白质差异的表达

目的 应用比较蛋白质组学方法筛选出肺结核患者发病免疫相关蛋白质,为阐明结核病的发病的免疫机制和诊断治疗提供理论依据.方法 收集10例肺结核患者和10名健康志愿者外周血单个核细胞(PBMC),采用双向凝胶电泳分离细胞总蛋白,运用Image Master 2D Elite 5.0图像分析软件识别差异表达的蛋白质点,应用基质辅助激光解析电离飞行时间质谱获取肽质量指纹图谱,检索数据库鉴定差异表达的蛋白质点,并对差异表达的蛋白质用Western blot进行验证.两组间比较采用秩和检验.结果 建立了重复性良好的肺结核患者和健康志愿者PBMC的双向凝胶电泳图谱,找出14个表达量有明显差异的蛋白质点并鉴定出共12种蛋白质,其中冠蛋白1A等10种蛋白在肺结核组中表达上调,普列克底物蛋白(PLEK)和Ras基因抑制蛋白1(RSU1)在肺结核组中表达下调;Western blot法验证PLEK在肺结核组表达下调,与双向凝胶电泳结果一致.结论 对肺结核患者PBMC的比较蛋白质组学研究鉴定出冠蛋白1A、PLEK等12种差异蛋白质,其中PLEK和冠蛋白1A可能通过对单核巨噬细胞吞噬作用的调节在肺结核发病中起着重要作用.     

江苏不同地区胃疾病相关幽门螺杆菌差异蛋白质的比较

目的:鉴定、比较江苏地区胃癌高、低发区胃癌与慢性胃炎相关幽门螺旋杆菌(Helicobacter pylori,H.pylori)差异表达蛋白质.方法:选取江苏省泰州市(胃癌高发区)和苏州市(胃癌低发区)胃癌和慢性胃炎患者胃镜下胃黏膜活检组织分离培养出的H.pylori,提取蛋白质后通过双向凝胶电泳进行分离,使用PDQuest软件对蛋白质点进行分析和比较,通过四极杆飞行时间电喷雾串联质谱对差异蛋白质点进行鉴定,并通过Mascot数据库检索.结果:所选H.pylori菌株总蛋白进行双向凝胶电泳后获得优质2-DE图谱,经Q-TOP鉴定为1号(巯基过氧化物酶)、3号(超氧物歧化酶)、4号(尿素酶α)、5号(核苷二磷酸激酶)、8号(分子伴侣dnaK)、9号(S-核糖基同型半胱氨酸裂解酶)、13号(无机焦磷酸酶)、14号(DNA引导的RNA聚合酶α)和16号(分子伴侣60×103)9个差异蛋白质点,其中3号、4号、8号和14号蛋白质点在胃癌菌株中均为高表达,1号和5号蛋白质点在慢性胃炎菌株中均为高表达,9号、13号和16号蛋白质点仅在一个地区存在差异表达.结论:胃癌高、低发区胃癌及相应地区慢性胃炎H.pylori蛋白质比较存在一定差异,但其中2/3的差异蛋白质是一致的,胃癌高发区H.pylori差异蛋白质高表达多于低发区,表明H.pylori在不同胃癌发病区致病机制相似,但在胃癌高发区作用强于低发区.     

吲哚美辛抗人结肠癌细胞系HCT116的功能蛋白质组研究

目的　探讨吲哚美辛 (IN)对人结肠癌细胞系HCT116蛋白质表达谱的影响及其抗结肠癌的作用机制。方法　应用固相 pH梯度双向凝胶电泳 (2 DE)技术分离IN治疗组和对照组HCT116细胞的总蛋白 ,银染显色 ,采用ImageScan扫描获取电子图像 ,ImageMaster 2D图像分析软件分析 2组的2 DE图谱并识别差异表达的蛋白质。应用基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)得到相应的肽质量指纹图谱 ,分析并鉴定差异蛋白质。结果　获得了背景清晰、分辨率高、重复性好的HCT116细胞系 2 DE图谱 ,对照组蛋白质点数为 12 99± 5 5 ,其匹配率为 94 .2 % ;IN治疗组蛋白质点数为 12 0 8± 4 7,其匹配率为 91.0 %。两组共有 4 5个差异蛋白质点 ,其中 ,治疗组有 9个蛋白质点表达上调 ,34个蛋白质点表达下调 ,2个蛋白质点仅在对照组表达。对差异蛋白质点进行了肽质指纹图分析发现 ,IN可通过BfL 1蛋白诱导HCT116细胞凋亡 ,并鉴定出一些与细胞凋亡、细胞周期及信号转导相关的蛋白质。结论　IN可通过BfL 1等多种途径诱导HCT116细胞的凋亡并抑制其增殖。     

类风湿关节炎患者外周血高迁移率族蛋白1表达

目的通过检测类风湿关节炎（RA）患者外周血单个核细胞（PBMC）、血浆中高迁移率族蛋白1（HMGB1）表达,为寻找治疗RA的新靶点提供依据。方法采集38例活动期RA患者、24例相对稳定期RA患者和20例健康对照者外周血。RT．PCR检测PBMC HMGB1mRNA表达,Western blot检测PBMC、血浆HMGB1蛋白表达。结果与相对稳定期RA患者、健康对照者相比,活动期RA患者PBMC HMGB1mRNA表达水平差异无统计学意义（F=1．23,P〉0．05）,而HMGB1蛋白表达水平下降（F=70．91,P〈0．01）,血浆HMGB1水平显著增高（P〈0．001）。相对稳定期RA患者与健康对照者之间差异无统计学意义（P〉0．05）。活动期RA患者血浆HMGB1水平与ESR（r．=0．478。P〈0．001）、C-反应蛋白（rs=0．574,P〈0．05）呈正相关。结论HMGB1与RA发病密切相关,并可能成为新的治疗靶点。     

Epstein-Barr virus load in the peripheral blood of patients with rheumatoid arthritis: accurate quantification using real-time polymerase chain reaction

OBJECTIVE: To determine whether patients with rheumatoid arthritis (RA) have elevated Epstein-Barr virus (EBV) load in their peripheral blood mononuclear cells (PBMCs) and whether it is correlated with the HLA-DR genes they express, we developed an accurate EBV DNA quantitative assay using real-time polymerase chain reaction (PCR) with fluorescent probes. METHODS: We studied the EBV DNA load in the PBMCs of 84 patients with RA, 69 normal controls, and 22 patients with rheumatic conditions other than RA. A 214-bp segment from the long internal repeat of EBV was amplified from 500 ng of PBMC DNA (150,000 cells) and quantified by real-time PCR with fluorescent probes. RESULTS: We demonstrated that in patients with RA, the EBV DNA load in PBMCs is increased almost 10-fold compared with that in normal controls. The EBV load is stable over time and is not obviously influenced by disease-modifying antirheumatic drugs or HLA-DR. CONCLUSION: Patients with RA have elevated EBV load in their peripheral blood.     

IL-2诱导类风湿关节炎与骨关节炎T淋巴细胞STAT3 STAT5酪氨酸磷酸化状态的比较

目的观察白细胞介素(IL)-2作用于类风湿关节炎(RA)与骨关节炎(OA)外周血T淋巴细胞前后,T淋巴细胞中STAT3及STAT5的酪氨酸磷酸化活化状态,并进行比较.方法从RA与OA患者的外周血中分离培养单个核细胞,继而纯化得到T淋巴细胞,静息后,用重组人IL-2刺激,在各时相裂解细胞,收获提取蛋白,进行Western blot分析.结果在静息后,RA与OA的外周血T淋巴细胞中STAT3与STAT5均处于极低水平磷酸化的状态;在IL-2作用后,T淋巴细胞中STAT3和STAT5发生时相性的酪氨酸磷酸化,而RA的T淋巴细胞磷酸化程度较OA显著增高.结论IL-2对RA患者T淋巴细胞的STAT3和STAT5过度激活,引起T淋巴细胞中IL-2信号传导的异常放大效应,可能在RA的发病过程中发挥重要作用.     

Interleukin-2 production and effect of thymosin fraction 5 on interleukin-2 production in rheumatoid arthritis

Interleukin-2 (IL-2) production and effects of thymosin fraction 5 (F5) on IL-2 production by peripheral blood mononuclear cells from patients with active rheumatoid arthritis (RA) were studied. Three patients with RA had elevated IL-2 production which was which was suppressed by F5. IL-2 production by peripheral blood mononuclear cells from 17 other patients with RA was similar to controls and was not affected by F5. Our results are consistent with hyperproduction of IL-2 by some patients with RA which is suppressed by F5.     

Different cytokine profiles in patients with chronic and acute reactive arthritis

OBJECTIVE: Analysis of cytokine production in patients with acute and chronic reactive arthritis (AcReA/ChrReA) in order to search for new treatment possibilities. METHODS: Cytokine production by peripheral blood and synovial fluid mononuclear cells (PBMCs/SFMCs) of 28 patients with AcReA, 27 patients with ChrReA, 26 patients with rheumatoid arthritis (RA) and 31 healthy controls was analysed by enzyme-linked immunosorbent assay (ELISA) and flow-cytometry. Production of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin (IL)-10 was measured by ELISA, while the percentages of TNF-alpha-, IFN-gamma- and IL-4-positive CD3+ cells were determined in the same groups of patients and healthy subjects using flow cytometry. RESULTS: Spontaneous TNF-alpha production observed in PBMCs of ChrReA, but not of AcReA, patients was significantly higher (P<0.001) than in healthy controls. The percentages of TNF-alpha-positive CD3+ blood cells in ChrReA exceeded that of RA patients and healthy controls (P<0.05 and P<0.001, respectively). Also, the percentages of IFN-gamma-positive CD3+ cells were significantly higher in peripheral blood and synovial fluid of ChrReA patients (P<0.05 and P<0.05, respectively) as compared with AcReA. In ChrReA spontaneous IL-10 production in PBMCs was similar to that observed in healthy controls, while in RA and AcReA the production of IL-10 was significantly increased (P<0.05 and P<0.05, respectively). IL-4 production was low in all study groups with no significant differences detected. CONCLUSIONS: High production of TNF-alpha and IFN-gamma detected in ChrReA supports the possible use of anti-TNF-alpha treatment in ChrReA.     

A proteomic study of peripheral blood mononuclear cells in systemic lupus erythematosus

Our objective was to analyze the changes in the protein expression profiles of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Peripheral blood was obtained from patients with SLE and healthy controls. 2-D gel electrophoresis was performed, and gels were silver-stained. Differentially expressed protein spots were detected, some of which were identified by MALDI-TOF spectrometry. Match rates of 71% +/- 4% and 72% +/- 4% were gotten for control and patient gels, respectively. 791 +/- 17 spots were detected for control gels and 781 +/- 17 for patient gels. Eleven protein spots were up-regulated, and 9 protein spots were down-regulated in patients with SLE. Five differentially expressed proteins were identified as immunoglobulin J chain, apolipoprotein A-IV precursor, calprotectin L1H and zinc finger protein subfamily 1A (all up-regulated) and glutathione S-transferase (down-regulated), some of which had previously been shown to play a potential role in the pathogenesis of SLE. We conclude there are significant changes in the 2-D maps of PBMCs in patients with SLE and applying this proteomic approach may be a useful way to gain novel insights into SLE.     

Relations between surface expression of the interleukin-2 receptor and release of the soluble form of the receptor in cultured mononuclear cells from patients with rheumatoid arthritis or systemic lupus erythematosus

The relationship between surface expression of the interleukin-2 receptor (IL-2R) and release of the soluble form of the receptor (sIL-2R or sCD25) was investigated with peripheral blood mononuclear cells (PBMCs) from individuals with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). The spontaneous release of sCD25 was significantly increased in PBMCs from RA patients and decreased in cells from SLE patients, compared with normal controls. However, the extent of sCD25 release from phytohaemagglutinin (PHA)-stimulated PBMCs did not differ between RA or SLE patients and normal controls. The serum concentration of sCD25 was significantly increased in SLE or RA patients compared with the normal controls. Whereas the surface expression of CD25 by unstimulated PBMCs did not differ among the three groups of subjects, this parameter was significantly reduced for PHA-stimulated PBMCs from RA patients relative to those from normal controls. The surface expression of CD25 showed a positive correlation with sCD25 release for PBMMCs from SLE patients under either basal or stimulated conditions. No such relation was apparent for cells from RA patients. The surface expression of IL-2R (CD122) under basal or stimulated conditions was significantly reduced in PBMCs from RA or SLE patients, compared with cells from normal controls. Thus, the increased concentration of sCD25 in the serum of individuals with these autoimmune rheumatic diseases may result from two different mechanisms: an increase in the spontaneous release of sCD25 in RA, and reduced clearance of this protein in SLE.     

活化的类风湿关节炎患者外周血单个核细胞Toll样受体4诱导Th17细胞分化

目的 研究类风湿关节炎(RA)患者外周血单个核细胞(PBMCs) Toll样受体4(TLR4)通过诱导Th17细胞分化参与RA疾病发生发展的意义.方法 采集22例RA患者和20名健康对照外周血,流式细胞术检测Th17细胞频率,脂多糖刺激PBMCs 2 d,实时荧光定量聚合酶链反应(RT-qPCR)检测PBMCs TLR4 mRNA水平,酶联免疫吸附试验(ELISA)法检测上清液白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α的含量.收集上清液与CD4+脐带血单个核细胞(CBMCs)培养3d,RT-qPCR检测CBMCsIL-17 mRNA水平.ELISA检测上清液IL-17含量.统计学处理采用成组t检验.结果 与健康对照组比较,RA患者Th17细胞频率[(0.53±0.14)％与(1.57±0.68)％]、TLR4 mRNA显著升高(P均＜0.01);PBMCs经脂多糖刺激后,RA患者TLR4 mRNA升高3.5倍,而健康对照组下降到0.11倍;RA患者PBMCs产生的IL-6、TNF-α较健康对照组明显增加(P＜0.01);RA患者PBMCs上清液诱导CD4+CBMCs IL-17 mRNA水平、IL-17含量与健康对照组相比均显著升高(P＜0.01);而未经脂多糖刺激,2组差异无统计学意义(P＞0.05).结论 RA患者PBMCs的TLR4表达及对脂多糖的刺激的反应性增加,且具有较强的诱导Th17细胞分化能力.