旋毛虫钙网蛋白对补体甘露糖结合凝集素途径活化的影响?

为研究旋毛虫感染的致病机制,探讨旋毛虫钙网蛋白对补体凝集素途径活化的影响,本文首先通过ELISA和Far western blot方法检测重组旋毛虫钙网蛋白(recombinant Trichinella spiralis calreticulin,rTs-CRT)与甘露糖结合凝集素(mannose binding lectin,MBL)的相互作用情况,随后采用ELISA法研究rTs-CRT对血清中的MBL以及重组MBL分子与甘露糖的结合的抑制作用,最后,研究这种抑制效果是否会抑制补体凝聚素途径的活化。ELISA及Far western blot结果证实rTs-CRT可结合MBL分子,且二者的结合可抑制血清以及重组MBL分子结合甘露糖。同时,这种抑制作用会减弱补体凝集素途径活化后C4b的沉积。结果表明,rTs-CRT可通过结合补体MBL抑制其识别甘露糖,进而减弱补体凝集素途径的活化。     

基于iTRAQ技术筛选妊娠糖尿病患者血清差异蛋白

目的 应用同位素标记相对和绝对定量（iTRAQ）联合液相色谱-串联质谱（LC-MS/MS）技术筛选和鉴定妊娠糖尿病（GDM）患者血清差异蛋白谱,探讨GDM的发病机制以期寻找潜在生物标志物。方法 收集2020年10月至2021年6月产检的孕晚期孕妇血样保存;追踪其妊娠结局,收集最终确诊为妊娠糖尿病及其正常对照组孕妇血清样本各4例,应用iTRAQ联合LC-MS/MS技术对样本进行鉴定,对差异蛋白进行生物信息学分析并构建蛋白互作（PPI）网络。结果 妊娠糖尿病及其对照组中共鉴定出215种差异表达蛋白,其中47种蛋白表达差异显著,上调31个,下调16个;生物信息学分析显示研究组中存在与脂质代谢、凝血级联激活、补体系统及炎性反应相关的蛋白表达异常;通过构建PPI网络发现差异表达蛋白主要分为：以APOB、APOE及APOM为代表的脂质代谢相关蛋白,且这些蛋白处于蛋白质互作网络的中心位置;以CRP、IL-6及C1R为代表的炎性因子及补体相关蛋白;以SERPING、F7及F9为代表的凝血相关蛋白,其结果与KEGG富集通路分析结果基本一致。结论 iTRAQ联合LC-MS/MS技术能有效地筛选出妊娠糖尿病...     

二维电泳和质谱技术鉴定肝细胞癌相关N-连接糖蛋白

目的 鉴定与肝细胞癌(HCC)发生发展相关的差异表达N-连接糖蛋白。 方法 采用刀豆凝集素A(ConA)、晶状体凝集素(LCH)、雪花凝集素(GNA)等3种植物凝集素组成的亲和层析柱,分别从肝永生化细胞系L02和HCC细胞系Huh7、PLC5、SNU449中纯化N-连接糖蛋白、二维电泳分析差异表达的蛋白质斑点,质谱、生物信息学等技术鉴定差异表达蛋白,Western blotting验证了转录调控肿瘤蛋白(TCTP)、上皮细胞黏附分子（EpCAM）和膜联蛋白A2（annexin A2）在肝永生化及HCC细胞和HCC及癌旁组织中的表达规律,体外侵袭实验检测TCTP沉默后对SNU449的侵袭力影响。 结果 质谱、生物信息学等技术共鉴定出42个差异表达的蛋白质分子/异质体（HCC细胞中高、低表达的蛋白/异质体分别为14个、28个）,代表32个蛋白质分子,其中的22个蛋白含有至少1个N糖基化位点（NetNGlyc 1.0预测）。这些差异表达的蛋白主要参与氧化还原内稳态维系、碳水化合物/能量代谢、糖酵解、抗凋亡等生物学过程。Western blotting检测表明,TCTP、EpCAM和annexin A2在6个肝癌细胞系PLC5、HepG2、SNU449、Huh7、HCC7721和SNU473及肝癌组织中表达显著升高,siRNA介导的TCTP沉默明显抑制了HCC细胞系SNU449的体外侵袭能力。 结论 TCTP、EpCAM和annexin可能参与了HCC发生发展过程,TCTP可能成为HCC靶向治疗的分子靶点之一。     

肺鳞癌患者和健康人血清的差异蛋白质组学研究

目的：比较肺鳞癌患者和健康人血清蛋白质组的差异,为筛选肺鳞癌血清分子标志物提供依据。方法：以5例健康人和5例I期肺鳞癌患者的血清为样本,采用15%的聚丙稀酰胺凝胶电泳(SDS-PAGE)分离血清蛋白质,Bandleader凝胶图像分析软件识别两种血清样本差异的蛋白质条带,电喷雾-四极杆-串联质谱（ESI-Q-TOF MS/MS）鉴定差异蛋白质条带中的蛋白质。Western印迹检测差异蛋白质结合珠蛋白-2（haptoglobin-2,HP-2)在肺鳞癌患者和健康人血清中的表达。结果：建立了健康人和肺鳞癌患者血清样品的一维凝胶电泳图谱,图像分析识别了4条差异蛋白质条带,质谱鉴定出29种非冗余的血清蛋白质。Western印迹证实了HP-2在肺鳞癌患者和健康人血清中的差异表达。结论：29种血清差异蛋白质为筛选肺鳞癌的血清分子标志物提供了实验依据。     

LPS诱导的A1型星形胶质细胞的能量代谢特征

目的：探讨在脂多糖（lipopolysaccharide,LPS）诱导下,小鼠皮层星形胶质细胞转化为A1毒性表型的同时,其能量代谢所发生的变化。方法：小鼠皮层星形胶质细胞培养8～9 d后,分为对照（control,CON）组和LPS组;采用CCK-8细胞增殖及毒性检测试剂盒检测不同LPS处理浓度及不同处理时间下的细胞活力;通过细胞免疫荧光染色技术检测胶质细胞原纤维酸性蛋白（glial fibrillary acidic protein,GFAP）的表达来鉴定星形胶质细胞的纯度;通过补体C3(complement component 3,C3)与GFAP细胞免疫荧光染色共定位,检测C3的表达水平;利用RT-qPCR技术检测C3、鸟苷酸结合蛋白2(guanylate-binding protein 2,GBP2)、S100钙结合蛋白A10(S100 calcium-binding protein A10,S100A10)、转谷氨酰胺酶1(transglutaminase 1,TGM1)及白细胞介素1β(interleukin-1β,IL-1β)的mRNA表达变化;采用Western blo...     

胰腺癌中MAT1蛋白的表达与其临床病理特征的关系

目的：研究MAT1蛋白在胰腺癌中的表达及其与胰腺癌多项临床病理特征的关系。 方法： 使用免疫组化首先分析了70例外科手术切除的胰腺癌标本、10例胰腺良性肿瘤、14例慢性胰腺炎和10例尸解来源正常胰腺组织标本中的MAT1蛋白表达。然后比较MAT1蛋白的表达与胰腺癌患者多项临床病理特征的关系，包括患者年龄、性别、癌灶部位、癌灶大小、组织学类型、分化程度和TNM分期。 结果： MAT1蛋白主要表达于胰腺癌癌细胞，也可见于成纤维细胞，棕黄色染色局限于细胞浆和核膜。75.7％（53/70）的胰腺癌组织中可见MAT1蛋白的表达，而正常对照标本中则未表达或仅微弱表达MAT1。MAT1在4种组织中的表达程度有显著差异（P<0.01）。其中MAT1在胰腺癌中的表达显著高于其它3种组织。进一步分析发现，MAT1蛋白表达与胰腺癌患者的TNM分期呈显著相关（P<0.05），但与其它临床病理特征无关。 结论： 在胰腺癌中表达明显上调的MAT1蛋白可能与胰腺癌的发生相关，并且MAT1蛋白是评估胰腺癌患者预后的一个有用的肿瘤标志物。     

系统性红斑狼疮肾炎血清差异蛋白质的筛选

目的:对比分析系统性红斑狼疮肾炎(LN)患者和健康人双重滤过血浆置换(DFPP)产物中的差异蛋白质,筛选与LN发生相关的血清蛋白质。方法:利用DFPP的方法分别对4例LN患者和4例健康人血浆进行过滤,将滤下的大分子蛋白混合物行双向电泳分离并银染显影,经图像分析筛选差异表达的蛋白点。应用QSTAR Pulsar I型串连飞行质谱鉴定差异表达的蛋白点,通过生物信息学比对,对其中有意义分子行血清浓度检测验证。结果:2组间筛选出差异蛋白点共317个,其中表达量差异达3倍以上的蛋白点69个,经串连质谱分析鉴定出包括人冷凝集素IgM抗体、免疫球蛋白λ轻链、谷胱甘肽-S-转移酶、结合珠蛋白前原蛋白、甲状腺素转运蛋白、载脂蛋白E、皮质类固醇结合球蛋白、玻连蛋白、角蛋白和转铁蛋白等十多个在SLE活动性肾炎血清中表达改变的蛋白分子。ELISA及RIA检测结果与双向电泳相符。结论:LN患者血清与健康人血清的蛋白表达谱有一定差异,这些差异分子可能成为LN诊断及预后判断的重要标志物。     

新的HCC血清标志物GP73在HCC中的表达及临床价值研究

目的研究新的原发性肝细胞癌（HCC）血清标志物高尔基体蛋白73（GP73）在HCC血清中的表达及临床价值。方法选取确诊为HCC者40例为HCC组;肝炎患者及肝硬化患者120例为肝病组;健康体检者120例为健康组。用双抗体夹心ELISA法检测三组血清GP73浓度;同时检测甲胎蛋白（AFP）及d．L．岩藻糖苷酶（AFU）作对比研究。结果HCC组血清中GP73的表达水平为（399．17±264．43）ng／ml,显著高于肝病组的（105．12±67．79）ng／ml和健康组的（66．51±46．2）ng／ml,差异均有统计学意义（P〈0．05）。GP73、AFP、AFU诊断HCC的敏感性分别为89．28％、62．5％和80．7％：特异性分别为87．1％、78．5％和68．7％,GP73敏感性及特异性均高于AFP和AFU。结论GP73是继AFP、AFU之后另一个新的优秀的诊断HCC血清标志物,具有重要临床价值。     

HSP27作为评价皮肤急性刺激性生物标志物的研究*

 目的：应用蛋白组学技术筛选用于评价化合物皮肤急性刺激性的特异性生物标志物。方法：选取2种欧盟列举的具有代表性的皮肤刺激性化合物十二烷基硫酸钠（SLS）、10-十一碳烯酸（UA）和1种非刺激性化合物3,3-二硫代二丙酸（DA）,严格按照体外评价皮肤刺激性替代方法的检测流程,使人体皮肤组织暴露于化合物15 min,然后裂解组织,提取蛋白,进行二维电泳;选择蛋白表达差异点,进行质谱鉴定,并用real-time RT-PCR和Western blotting验证差异蛋白的表达。结果：在鉴定的8个蛋白点中,SLS和UA刺激使人体皮肤热休克蛋白27（HSP27）的表达显著上调,与Western blotting结果一致。结论：HSP27有可能成为评价皮肤刺激性体外替代方法体系的候选生物标志物,其表达强度与刺激强度的关系需进一步探讨。     

基于iTRAQ技术的早发型重度子痫前期患者胎盘组织定量蛋白质组学研究

目的 应用同位素标记相对和绝对定量（iTRAQ）联合液相色谱-串联质谱（LC-MS/MS）技术筛选和鉴定早发型重度子痫前期（EOSP）患者胎盘组织差异蛋白,探讨EOSP的发病机制并寻找早期生物标志物。方法 收集2020年5月至2021年5月于文昌市人民医院产检并分娩的孕妇,收集其孕早期血样保存;追踪其妊娠结局,收集最终诊断为EOSP及其正常对照组孕妇胎盘组织各4例,应用iTRAQ联合LC-MS/MS技术对样本进行鉴定,利用生物信息学分析得到的差异表达蛋白;应用酶联免疫吸附测定（ELISA）对孕早期血样中的关键蛋白进行验证。结果 EOSP及其对照组中共鉴定出278种差异表达蛋白,其中37种蛋白表达差异显著,上调21个,下调16个;生物信息学分析显示EOSP中存在与脂质代谢、凝血级联激活及补体系统相关的蛋白表达异常;利用ELISA法验证两组患者孕早期血清关键蛋白基质金属蛋白酶2(MMP2)水平,数据显示EOSP组中MMP2水平较对照组显著降低,差异有统计学意义（P<0.05）。结论 iTRAQ联合LC-MS/MS技术能有效地筛选出EOSP的胎盘组织差异表达蛋白,MMP2有可能成为早期...     

The acute phase protein response in patients receiving subcutaneous IL-6.

IL-6, tumour necrosis factor-alpha (TNF-alpha) and IL-1 are thought to be the key mediators of the acute phase response although much of the evidence is based on in vitro studies. It is not clear to what extent each of the acute phase proteins are regulated in vivo by each of these cytokines. The aim of this study was to examine the effects of IL-6 treatment in eight patients with cancer on the concentrations of an extensive range of positive and negative acute phase proteins. It was part of a larger investigation to assess the value of IL-6 in the management of chemotherapy-induced thrombocytopenia. IL-6 was administered by a daily subcutaneous injection for 7 days at a dose level of 1, 3, or 10 micrograms/kg/day. Increases in the positive acute phase proteins, serum amyloid A, C-reactive protein, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, haptoglobin, alpha 1-antitrypsin, fibrinogen, complement component C3, and caeruloplasmin, were observed, with the greatest incremental changes and fastest responses being seen for C-reactive protein and serum amyloid A protein. The negative acute phase proteins transferrin, transthyretin and retinol binding protein all fell to a nadir within 48-96 h after the first IL-6 injection. Increases in complement component C4 were only found in two patients, which may be related to the increase in circulating TNF-alpha concentrations found only in these patients. This study has therefore shown that IL-6 is capable of causing changes in the majority of acute phase proteins in vivo. Although secondary induction of TNF-alpha was not observed in the majority of patients examined, it is still possible however that other cytokines involved in regulation of the acute phase response, such as IL-1, may have been induced and contributed to the overall response.     

Serum and tissue level of insulin-like growth factor-I (IGF-I) and IGF-I binding proteins as an index of pancreatitis and pancreatic cancer

Previously we have found deregulation of collagen metabolism in human pancreatitis and pancreatic cancer tissues. Insulin-like growth factor-I (IGF-I) is known to stimulate collagen biosynthesis through interaction with IGF-I receptor. IGF-I binding proteins (BPs) regulate the activity of IGF-I. We investigated whether serum and tissue IGF-I and IGF-BPs as well as tissue IGF-I receptor expression may reflect disturbances of collagen metabolism in patients with pancreatitis and pancreatic cancer. In pancreatitis tissue, a significant increase in IGF-I and IGFBP-3 content was accompanied by a distinct increase in IGF-I receptor expression, compared to control pancreas tissue. In contrast, serum from patients with pancreatitis did not show significant increases in IGF-I and IGFBP-3 levels, however, significant increases in IGFBP-1 level (2.5 fold). Moreover, a distinct decrease in radioactive IGF-binding to the BPs, compared to control serum, was found. Pancreatic cancer tissue and serum of patients with pancreatic cancer showed significant increases in IGF-I, IGFBP-3 and IGFBP-1 content, accompanied by dramatic increases in IGF-I tissue receptor expression, compared to controls. In serum of patients with pancreatic cancer distinct increases in radioactive IGF-binding to 46 kDa BP, compared to control serum, were observed. The data suggest that disturbances in tissue collagen metabolism during pancreatic diseases may result from deregulation of IGF-I homeostasis and that elevated serum levels of IGF-I, IGFBP-3 and IGFBP-1 may serve as markers of pancreatic cancer.     

APP,APOE, complement receptor 1, clusterin and PICALM and their involvement in the herpes simplex life cycle

The major Alzheimer's disease susceptibility genes (APOE, clusterin, complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein, PICALM) can be implicated directly (APOE, CR1) or indirectly (clusterin and PICALM) in the herpes simplex life cycle. The virus binds to proteoliposomes containing APOE or APOA1 and also to CR1, and both clusterin and PICALM are related to a mannose-6-phosphate receptor used by the virus for cellular entry and intracellular transport. PICALM also binds to a nuclear exportin used by the virus for nuclear egress. Clusterin and complement receptor 1 are both related to the complement pathways and play a general role in pathogen defence. In addition, the amyloid precursor protein APP is involved in herpes viral transport and gamma-secretase cleaves a number of receptors used by the virus for cellular entry. APOE, APOA1 and clusterin, or alpha 2-macroglobulin, insulysin and caspase 3, which also bind to the virus, are involved in beta-amyloid clearance or degradation, as are the viral binding complement components, C3 and CR1. There are multiple ways in which the products of key susceptibility genes might be able to modify the viral life cycle and in turn the virus interacts with key proteins involved in APP and beta-amyloid processing. These interactions support a role for the herpes simplex virus in Alzheimer's disease pathology and suggest that antiviral agents or vaccination might be considered as viable therapeutic strategies in Alzheimer's disease.     

Tissue microarray methodology identifies complement pathway activation and dysregulation in progressive multiple sclerosis

The complement pathway has potential contributions to both white (WM) and grey matter (GM) pathology in Multiple Sclerosis (MS). A quantitative assessment of complement involvement is lacking. Here we describe the use of Tissue MicroArray (TMA) methodology in conjunction with immunohistochemistry to investigate the localization of complement pathway proteins in progressive MS cortical GM and subcortical WM. Antibodies targeting complement proteins C1q, C3b, regulatory proteins C1 inhibitor (C1INH, complement receptor 1 (CR1), clusterin, factor H (FH) and the C5a anaphylatoxin receptor (C5aR) were utilised alongside standard markers of tissue pathology. All stained slides were digitised for quantitative analysis. We found that numbers of cells immunolabelled for HLA‐DR, GFAP, C5aR, C1q and C3b were increased in WM lesions (WML) and GM lesions (GML) compared to normal appearing WM (NAWM) and GM (NAGM), respectively. The complement regulators C1INH, CR1, FH and clusterin were more abundant in WM lesions, while the number of C1q+ neurons were increased and the number of C1INH+, clusterin+, FH+ and CR1+ neurons decreased in GM lesions. The number of complement component positive cells (C1q, C3b) correlated with complement regulator expression in WM, but there was no statistical association between complement activation and regulator expression in the GM. We conclude that TMA methodology and quantitative analysis provides evidence of complement dysregulation in MS GML, including an association of the numerical density of C1q+ cells with tissue lesions. Our work confirms that complement activation and dysregulation occur in all cases of progressive MS and suggest that complement may provide potential biomarkers of the disease.     

Th22细胞相关因子及补体蛋白在药疹患者治疗前后血清中的表达及意义

探讨 Th22细胞相关因子及补体蛋白在药疹患者治疗前后血清中的表达水平及其意义.方法 临床选择2015年7月至2016年4月本院收治的药疹患者45例为观察组,同期选择体检健康者45例为对照组.观察组均进行钙剂、维生素C、抗组胺药物、支持疗法等治疗.观察组分为治疗前和治疗后两组.通过流式细胞术多重蛋白定量技术(CBA)检测各组血清C5a,C4a和C3a等补体蛋白水平;通过酶联免疫吸附试验(ELISA)测定各组血清TNF-α,白细胞介素13(IL-13)和白细胞介素22(IL-22)等细胞因子的水平.结果 治疗前,C5a,C4a和C3a以及TNF-α,IL-13,IL-22均明显高于对照组(P＜0.05).治疗后,这些指标均显著低于治疗前水平(P＜0.05),但仍高于对照组(P＜0.05).结论 Th22细胞相关因子和补体激活在药疹快速发展中起重要作用.IL-22参与补体蛋白的调控.     

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.     

Identification of specific serum proteins synthesized de novo by monolayer cultures of glandular cells of gestational endometrium

Monolayer cell cultures (n = 3) of glandular epithelium of gestationalendometrium obtained from three apparently healthy women undergoingelective termination of pregnancy (7–9 weeks gestation)were established. De novo synthesis of eight serum proteins(albumin, alpha₁-antitrypsin, cerulo-plasmin, beta-lipoprotein,alpha₂-macroglobulin, fibronectin and complement factors C3and C4) was demonstrated by the incorporation of radiolabelledsubstrate ([³⁵-S]methionine) employing autoradiography (AR)in combination with crossed immunoelectrophoresis (XIE), referredto as ARXIE, and line immunoelectrophoresis (LIE), referredto as ARLIE. By contrast, there was no evidence for de novosynthesis of IgA, haptoglobin and orosomucoid. Our findingssuggest that the gestational endometrium may contribute to theproduction of several proteins considered to be synthesizedand secreted mainly by the liver and reticulo-endothelial system.The simple techniques used here to identify the de novo synthesisof human serum proteins could be applied to investigate proteinsynthesis by a wide range of tissues and cells     

A high incidence of C9 deficiency among healthy blood donors in Osaka, Japan

By the use of sucrose gelatin veronal buffer (SGVB), a simple screening test was developed by us to detect sera with low complement activity, including C9-deficient sera. Using this screening test, we were able to identify sera with low complement activity including C9-deficient sera among a large number of samples. Further examinations, estimation of the protein concentration of C9, C4, C3, etc., enabled classification of serum with low complement activity into C9-deficient serum, serum deficient in the other components, and serum with low complement activity caused by non-specific activation of complement through the classical pathway by low temperature in vitro. Among 145,640 sera from Osaka donors, 138 sera were found to be deficient in C9 by these methods. The whole complement activity (CH50) of the 138 sera was 13.1 +/- 3.0 U/ml. The C9 protein in these sera was undetectable, not only by the single radial immunodiffusion method, but also by the sensitive ELISA method. C9 activities in these sera were less than 0.1% of the level in pooled normal human serum. These findings and the family studies revealed that 138 blood donors unquestionably had a hereditary C9 deficiency. The incidence of C9 deficiency among Osaka donors was calculated to be 0.095%.     

Presence of plasma complement regulatory proteins clusterin (Apo J) and vitronectin (S40) on circulating immune complexes (CIC)

The complement regulatory (CR) proteins clusterin and vitronectin bind to the membrane attack complex (MAC) and thus prevent cytolysis. In this report, we demonstrate the presence of both of these CR proteins on MAC bound to circulating immune complexes (CIC). We measured the amount of clusterin and vitronectin on MAC in plasma, also referred to as soluble MAC (SMAC), as well as on MAC bound to CIC (MAC-CIC), using antibody directed to polymerized C9 in systemic lupus erythematosus (SLE) patients. We observed a strong correlation among the quantities of SMAC and MAC-CIC. The amount of both clusterin and vitronectin associated with MAC-CIC was two- to threefold higher in comparison to the SMAC. Patients with high levels of clusterin and vitronectin demonstrated renal involvement. We hypothesize that these complement regulatory proteins besides regulating the insertion of MAC play other critical roles, in disease pathogenesis.