重症肌无力患者胸腺自身免疫相关基因表达的研究

目的探讨MG患者和正常对照者的胸腺组织炎症反应与自身免疫性疾病相关基因的表达情况。方法取MG患者（MG组）和正常对照者（对照组）的胸腺组织标本，提取总RNA，反转录合成cDNA后，体外转录合成生物素标记的cRNA与基因芯片进行杂交，通过化学发光法检测基因表达，比较两组胸腺组织基因表达的差异。结果MG组与对照组比较，有61条基因存在显著性差异，其中表达上调33条，下调28条，这些基因包括炎症相关趋化因子基因、细胞因子及其受体基因、与细胞因子产生与代谢有关的基因、细胞因子及其受体相互作用相关的基因、炎症反应相关基因以及体液免疫相关基因等。结论芯片筛选MG患者胸腺自身免疫相关基因表达与正常对照组存在显著差异。     

cDNA微阵列对不同时期胶原诱导性关节炎大鼠PBMC基因表达谱的初步研究

目的 研究胶原诱导性关节炎（collagen induced arthritis，CIA）大鼠不同时期外周血单个核细胞（peripheral blood mononuclear cells，PBMC）基因表达谱，寻找与疾病炎症相关的特异表达基因，初步探讨类风湿关节炎（rheumatoid arthritis，RA）的发病机制。方法 以含有96个基因的功能基因组芯片研究CIA大鼠发病早期、高峰期和后期及正常大鼠的PBMC基因表达谱。结果 cDNA微阵列检测结果显示，CIA早期差异表达基因（〉2或〈0．5）有19个，高峰期25个，后期17个以及三个时期持续表达的上调基因有10个。差异表达基因主要涉及白细胞介素及其受体、趋化因子及其受体、转化生长因子配体及受体等。结论 CIA不同时期基因的差异表达可能与RA病情的持续进展有关，为进一步探讨细胞因子在RA的作用提供理论依据。     

基因芯片技术研究原发性干燥综合征患者唇腺基因表达

目的应用基因芯片技术筛选原发性干燥综合征（SS）患者与健康对照唇腺组织中的差异表达基因，探讨其可能的发病机制。方法实验组和对照组各3例，分别制成eRNA探针，与基因芯片杂交，筛选出差异表达基因。结果实验组与对照组显著差异表达基因181个，其中上调表达128个，下调表达53个。涉及细胞因子及受体相关基因、凋亡相关基因等。结论SS的发生发展受多种基因调控，差异基因表达谱的建立较全面地反映了SS的分子生物学概貌。     

老年大鼠下丘脑-垂体-肾上腺轴炎性衰老相关基因表达特征

目的探讨炎性衰老的炎性细胞因子网络机制。方法清洁级健康雄性SD大鼠分为4月龄（4m）和24月龄（24m）二组,每组各10只。分别取各组大鼠下丘脑、垂体和肾上腺组织,应用炎症细胞因子与受体基因芯片进行基因表达检测,绘制基冈表达谱,筛选有表达差异的基因。结果与4m组相比,24m组下丘脑、垂体和肾上腺组织中表达上调的促炎性反应细胞因子与受体基因分别有6个、7个和9个,表达下调的抗炎细胞因子与受体基因分别有3个、4个和3个（P〈0．05）。三种组织中表达都上调的促炎性反应细胞因子与受体基因有白介素、肿瘤坏死因子和趋化因子。结论老年大鼠下丘脑－垂体－肾上腺轴组织存在促炎性细胞因子基因表达上调,导致促－抗炎性细胞因子网络和促－抗炎性反应体系平衡失调,这可能是炎性衰老中出现高促炎性反应状态的原因和炎性衰老发生的新机制。     

抗环瓜氨酸肽抗体阳性和阴性类风湿关节炎患者外周血单个核细胞基因表达谱差异的研究

目的 运用基因芯片技术研究抗环瓜氨酸肽(CCP)抗体阳性和阴性类风湿关节炎(RA)患者外周血单个核细胞(PBMC)基因表达谱差异,探索差异存在的基因表达基础.方法 提取各5例抗CCP抗体阳性、阴性RA患者、健康人PBMC中总RNA,运用Ⅲumina基因表达谱芯片分析每个样本47231个基因变化.结果 与健康对照组相比,抗CCP抗体阳性和阴性RA患者有88个差异表达基因,其中上调表达基因51个,下调表达基因37个,这些差异表达基因主要涉及细胞因子、细胞凋亡、细胞周期、细胞因子、信号转导、趋化因子等.抗CCP抗体阳性和阴性RA患者之间有20个基因表达差异有统计学意义,其中上调表达基因9个,下调表达基因11个,这些差异表达基因主要涉及传递蛋白、转录调控、信号转导、代谢、细胞周期等.结论 抗CCP抗体阳性和阴性RA患者之间存在差异表达基因,筛选到的差异基因如杀伤细胞免疫球蛋白受体基因、干扰素诱导基因、几丁质酶家族成员CHI3L1等为进一步研究RA发病机制及发现新治疗靶标提供重要线索.     

应用基因芯片技术筛选胰腺癌相关基因

目的应用基因芯片技术筛选胰腺癌相关基因。方法将14000种人类基因PCR产物按微矩阵排列点样于化学涂层的载玻片上，制成基因芯片。按一步法抽提4例胰腺癌和癌旁正常胰腺组织的总RNA，将等量的RNA分别逆转录合成荧光分子掺人的cDNA一链作探针，混合后杂交上述基因芯片。经严格洗片后用ScanArray 4000扫描仪扫描芯片荧光信号图像，每点上两种荧光信号的强度分别代表Cy5-dCTP和Cy3-dCTP的量，获得的荧光信号图像用计算机分析。结果按差异显著性标准，从14000个基因中筛选出在胰腺癌组织中共同差异表达基因189条，其中已知基因101条，新基因88条。在筛选出的已知基因中，有50条表达上调，51条表达下调。结论基因芯片技术的肿瘤基因表达谱分析能够高通量筛选胰腺癌相关基因。并高效对基因功能进行研究。胰腺癌基因表达谱的分析有助于认识肿瘤发病机制。     

应用基因芯片技术对系统性红斑狼疮患者外周血基因表达谱的初步研究

目的 研究系统性红斑狼疮（SLE）患者与正常对照外周血的基因表达谱的改变，以探讨SLE发病机制，诊断及鉴别诊断，以及基因功能的研究。方法 首先从外周血提取总RNA，反转录合成单链，双链cDNA后，体外转录合成生物素标记的cRNA与基因芯片进行杂交，再通过抗原抗体反应机制标记上荧光染料Cy3后，使用芯片扫描仪进行图像扫描。使用分析软件进行表达差异和聚类分析。结果 在3000多个基因点中，与正常对照相比较，鉴定出94个基因存在表达差异相关基因，其中有33个基因表达上调，61个基因表达下调。这些表达差异基因有细胞因子及受体，转录相关基因，凋亡相关基因，生长分化相关基因，信号转导相关基因，细胞周期相关基因，电子传递和氧化相关基因，转移酶相关, 酶相关基因等。聚类分析结果显示不同的疾病以及不同临床表面的同一疾病其外周血的基因表达谱是有差异的，可以用于疾病的诊断和鉴别诊断。基因聚类发现，根据表达谱聚类在一起的基因存在功能上的相似性，可以用来发现已知基因的免疫相关功能。结论 该基因芯片技术方法有较高的重复性和稳定性，能有效地进行SLE致病基因的筛选，更好地理解SLE发生的分子机制，诊断及鉴别诊断，以及基因功能的研究。     

采用人基因表达谱芯片筛选肺癌转移相关基因的初步研究

目的应用基因芯片技术分析人高、低转移肺巨细胞癌细胞株的基因差异表达谱，筛选与肺癌转移相关基因。方法提取人高转移肺巨细胞株95D和人低转移肺巨细胞株95C的mRNA，通过逆转录，制备分别用cy5-dUTP和cy3-dUTP进行标记的cDNA探针，并与芯片杂交。经过ScanArray3000扫描仪扫描，获取图象及对杂交信号进行数据分析，筛选出95D和95C细胞表达差异的基因谱，并对其中部分基因进行RT—PCR分析、验证。结果在所检测95C和95D细胞中，466条基因有表达差异，其中具有同-Genebank量的有108对，上调基因47对，下调基因61对。对其中KIAA1108、PGR1、JWA、S182、Jab1部分差异表达基因，经RT-PCR技术验证，结果与基因芯片基本符合。结论肺癌转移过程与多种基因作用有关，基因芯片技术可为肺癌转移相关基因的筛选，提供有效方法。     

原发性干燥综合征患者细胞因子途径、TOLL样受体信号途径相关基因表达的初步探讨

目的筛选原发性干燥综合征（pSS）患者的外周血单个核细胞的细胞因子途径、TOLL样受体信号途径的差异表达基因,并探讨其在发病机制中的作用。方法取3例初次发病的pSS患者及3例健康对照者的外周血单个核细胞,提取总RNA,反转录并进行体外扩增,分别用Cy5及Cy3荧光标记pSS组及对照组的cDNA样品,与寡核苷酸基因芯片进行杂交,运用LuxScan5．0图像分析软件将芯片图像信号转化为数字信号,然后以Cy5／Cy3的比值数据进行分析（SAM软件）寻找差异表达基因,并对差异表达基因用分子功能注释系统（MAS）进行处理。结果pSS组与对照组外周血单个核细胞中的细胞因子途径相关基因、TOLL样受体信号途径相关基因的表达差异有统计学意义（P〈0．01）。以上基因途径中基因TNFSF13B、CD40、TNFRSF7、CD86表达上调,基因FAS、CD40LG、CD80表达下调。结论pSS患者细胞因子途径和TOLL样受体信号途径的TN—FSF13B、CD40、TNFRSF7、CD86、FAS、CD40LG和CD80多个基因的异常表达提示pSS患者B细胞的激活、分化、凋亡和免疫耐受等多方面功能的异常,而且pSS的发病可能是多基因调控异常的结果,这些基因可能成为新的治疗靶点。     

运用基因芯片初步研究类风湿关节炎患者外周血单个核细胞基因表达谱特征

目的运用基因芯片技术研究类风湿关节炎(RA)基因表达谱特征,从全基因组角度探讨RA相关致病基因。方法提取5例活动期RA患者和3名健康对照者外周血单个核细胞(PBMC)中总RNA,Illumina寡核苷酸基因芯片分析每个样本的48 000个基因变化。反转录-聚合酶链反应(RT-PCR)验证芯片结果。结果RA患者与健康对照者相比2倍上调基因122个,2倍下调基因29个。分子及生物功能分析显示上调表达的基因涉及传递蛋白、蛋白翻译合成、代谢、转录调控、信号转导、趋化因子、细胞周期和凋亡相关基因等种类。结论RA涉及多环节的病理过程,基因芯片技术有利于进一步揭示RA分子机制,发现新的治疗靶标。     

TNF Revisited: Osteoprotegerin and TNF-related Molecules in Heart Failure

The pathophysiological role of tumor necrosis factor (TNF) in myocardial failure has been extensively examined in experimental and clinical studies. Recent studies suggest that other members of the TNF/TNF receptor superfamily (TNFSF/TNFRSF) also may play a pathogenic role in chronic HF. TNF ligands, and in particular members of the TNFRSF, are expressed by a wide variety of cells, including myocardial cells. By activating the nuclear factor-κB (NF-κB) and death-related pathways, TNF ligands can induce a variety of effects within the myocardium, including apoptosis, hypertrophy, inflammation, and extracellular matrix remodeling. Among several TNFSF members that have been shown activated in HF, the OPG/RANK/RANKL (osteoprotegerin/receptor activator of NF-κB/RANK ligand) axis may be of importance in the pathogenesis of this disorder through different mechanisms. In this paper, we revisited the role of TNFSF/TNFRSF in the pathophysiology of HF, possibly representing new targets for therapy as well as new biomarkers in this disorder.     

A novel mutation (T61I) in the gene encoding tumour necrosis factor receptor superfamily 1A (TNFRSF1A) in a Japanese patient with tumour necrosis factor receptor-associated periodic syndrome (TRAPS) associated with systemic lupus erythematosus

OBJECTIVE: To identify potential mutations in the tumour necrosis factor receptor superfamily 1A gene (TNFRSF1A) in a Japanese female patient with recurrent fever complicated by systemic lupus erythematosus (SLE), and in her family members. METHODS: DNA sequencing of exons 1-10 of the TNFRSF1A gene was performed to determine mutations that might be associated with the tumour necrosis factor receptor-associated periodic syndrome (TRAPS). Moreover, the TNFRSF1A gene was examined in Japanese patients with autoimmune diseases, including SLE, rheumatoid arthritis (RA), mixed connective tissue disease (MCTD) and Beh?et's disease, and in healthy Japanese controls. Enzyme-amplified sensitivity immunoassay (EASIA) analysis was used to assess serum levels of TNF, the 55-kDa TNF receptor (TNFRSF1A) and the 75-kDa TNF receptor (TNFRSF1B). Membrane TNFRSF1A expression was analysed on the surface of peripheral blood mononuclear cells by flow cytometry. RESULTS: A novel mutation, a heterozygous C to T transition in exon 3 which substitutes an isoleucine for a threonine at position 61 (T61I) was detected in the TNFRSF1A gene derived from the genomic DNA of a Japanese female TRAPS patient. Two nieces and one nephew, all with a similar clinical phenotype, also possessed the same TNFRSF1A mutation. We further demonstrated the same mutation in five of 60 SLE patients (8.3%) and in five of 120 healthy individuals (4.2%), with no significant differences. Although high titres of serum TNF and soluble TNFRSF1B protein were observed in this patient, low titres of soluble TNFRSF1A protein were detected. However, a defect in TNFRSF1A shedding in vitro was not observed in monocytes derived from this patient. CONCLUSION: This is the first report of a TRAPS patient associated with SLE with a novel TNFRSF1A mutation (T61I).     

B-cell maturation antigen (BCMA) activation exerts specific proinflammatory effects in normal human keratinocytes and is preferentially expressed in inflammatory skin pathologies

TNFα is known to be expressed in human skin, regulating immune-related responses. Here we report that human normal skin keratinocytes express the members of the TNF superfamily members A proliferation-inducing ligand (APRIL; TNFSF13), B cell-activating factor (BAFF; TNFSF13B), and their receptors, B cell maturation antigen (BCMA; TNFRSF17) and transmembrane activator, calcium-modulator, and cyclophilin ligand interactor (TACI; TNFRSF13B), in a distinct spatial pattern. Our data show a differential expression of these molecules within epidermal layers and skin appendages, whereas the BAFF-specific receptor BAFFR (TNFRSF13C) is absent. Importantly, APRIL and BCMA but not BAFF or TACI are up-regulated in inflammatory skin lesions of psoriasis and squamous cell carcinomas. To explore the functional significance of this system in the skin, we assayed these receptors and ligands in cultured primary keratinocytes and HaCaT cells. We show that both cell types express BAFF, APRIL, BCMA, and TACI. Furthermore, APRIL and/or BAFF trigger nuclear factor-κB activation and IL-6 and granulocyte macrophage colony-stimulating factor (GM-CSF) expression through functional BCMA receptors, an activation inhibited by anti-BCMA short hairpin RNA. However, BAFF and/or APRIL do not induce IL-8 or TNFα production. Our data advance BCMA as an inflammation-related TNFSFR member in keratinocytes, of potential importance in the management of inflammatory skin conditions.     

Association of TNFRSF4 gene polymorphisms with essential hypertension

BACKGROUND: Essential hypertension is a complex disorder that results from the interaction of a number of susceptibility genes and environmental factors. The TNFRSF4 (tumor necrosis factor receptor superfamily, member 4) gene was one of the genes that showed altered renal expression in long-term salt loading in mice. Moreover, association of the TNFRSF4 and TNFSF4 (tumor necrosis factor (ligand) superfamily, member 4) genes with myocardial infarction was recently reported. Since essential hypertension is a well-known risk factor for myocardial infarction, we hypothesized that TNFRSF4 could be a susceptibility gene for essential hypertension. METHODS: We performed a case-control study of TNFRSF4 in two independent population. RESULTS: Extensive investigation of single nucleotide polymorphisms of the entire gene suggested that it resided in one linkage disequilibrium block, and four single nucleotide polymorphisms in the 5' flanking region sufficiently represented major haplotypes. In the combined population, the frequency of the most frequent haplotype, C-C-A-A, was significantly lower (P = 8.07 x 10(-5)) and that of the second most frequent haplotype, C-T-G-A, was significantly higher (P = 6.07 x 10(-4)) in hypertensive subjects than in control subjects. This difference was observed only in female patients. The C-T-G-A haplotype showed a lower promoter activity than other haplotypes, suggesting a relationship with disease susceptibility. CONCLUSION: Our results suggest that TNFRSF4 is a female-specific susceptible gene for essential hypertension.     

心力衰竭患者细胞因子网络及内源性调控的变化

目的了解细胞因子网络调控在心力衰竭发生、发展中的病理作用.方法采用双抗体夹心ELJSA法检测52例不同程度充血性心力衰竭病人和30例正常人肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、可溶性肿瘤坏死因子受体Ⅰ(sTNF-RⅠ)和转化生长因子β(TGF-β)的血浓度.结果心力衰竭组与正常组比较,血TNF-α、IL-6、sTNF-RⅠ明显增高,TGF-β显著降低,TGF-α/sTNF-RⅠ和TNF-α/TGF-β显著增高,尤其以心功能Ⅳ级组明显,TNF-α与IL-6和sTNF-RⅠ呈显著正相关,而与TGF-β呈显著负相关(P＜0.01).结论严重心力衰竭时存在细胞因子网络平衡紊乱,炎症性细胞因子增加、抗炎症性细胞因子相对或绝对不足,可能参与了心力衰竭的发生和发展.     

Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure

OBJECTIVE: Increasing evidence supports a role for inflammation in chronic heart failure (CHF). However, the source and the mechanism for this immune activation are unknown. To address this issue we investigated the gene expression of cytokines and the surface expression of activity markers in T-cells and monocytes from CHF patients and healthy controls. METHODS: Gene expression of cytokines was analysed by real-time RT-PCR and activation markers by flow cytometry in 14 CHF patients and nine healthy controls. Surface expression of activation markers for T-cells and monocytes were analysed by flow cytometry. RESULTS: T-cells from CHF patients showed enhanced gene expression of chemokines, ligands in the tumor necrosis factor superfamily, as well as the inflammatory cytokines interferon-gamma and interleukin-18 with similar pattern in ischemic (n=5) and idiopathic cardiomyopathy (n=9). In contrast, no differences in cytokine gene expression were found comparing monocytes from CHF patients and controls. Moreover, T-cells from CHF patients had enhanced surface expression of the activation markers CD69 and CD25, while there was no upregulation of the monocyte activation marker CD32 in these patients. CONCLUSION: T-cells may be a part of the inflammatory response during CHF independent of the etiology of the disorder. Intervention preventing unwanted T-cell activation could represent a new target in the treatment of CHF.     

Comment: the methionine 196 arginine polymorphism in exon 6 of the TNF receptor 2 gene (TNFRSF1B) is associated with the polycystic ovary syndrome and hyperandrogenism

Inflammatory cytokines such as TNF alpha may play a role in the pathogenesis of common metabolic disorders, including hyperandrogenism and the polycystic ovary syndrome (PCOS). The TNF receptor 2 mediates most of the metabolic effects of TNF alpha. In the present study, we have evaluated serum soluble TNF receptor 2 levels, and several common polymorphisms in the TNF receptor 2 gene (TNFRSF1B), in women presenting with PCOS or hyperandrogenic disorders. Initial studies included 103 hyperandrogenic patients (42 presenting with PCOS) and 36 controls from Spain. The 196R alleles of the M196R (676 T-->G) variant in exon 6 of TNFRSF1B, which is in linkage disequilibrium with a CA-repeat microsatellite polymorphism in intron 4 of TNFRSF1B, tended to be more frequent in hyperandrogenic patients than in controls (P = 0.056), reaching statistical significance when the analysis was restricted to include only PCOS patients (P < 0.03). Extended analysis including another 11 hyperandrogenic patients from Spain and 64 patients and 29 controls from Italy confirmed the association between 196R alleles of the M196R variant and hyperandrogenic disorders (P < 0.05), which was maintained when restricting the analysis to PCOS patients (P < 0.02). On the contrary, the 3'-untranslated region (exon 10) variants 1663 G-->A, 1668 T-->G, and 1690 T-->C were not associated with hyperandrogenism. The soluble TNF receptor 2 levels were not different between patients and controls but were increased in obese subjects, compared with lean individuals, and were affected by the interaction between the 1663 G-->A and 1668 T-->G variants in the 3'-untranslated region of TNFRSF1B. The TNFRSF1B genotype did not influence any clinical or biochemical variable related to hyperandrogenism or insulin sensitivity and was not associated with obesity, both in hyperandrogenic patients and healthy controls considered separately. In conclusion, the M196R (676 T-->G) variant in exon 6 of TNFRSF1B is associated with hyperandrogenism and PCOS, further suggesting a role for inflammatory cytokines in the pathogenesis of these disorders.     

Cytokine network in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy

Inflammatory cytokines may play a pathogenic role in the development of congestive heart failure (CHF). Elevated circulating levels of inflammatory cytokines have been reported in CHF, but most studies have focused on only a few cytokine parameters. However, the activity of these cytokines are modulated by soluble cytokine receptors and cytokines with anti-inflammatory activities, and in the present study several of these interacting factors were examined simultaneously in 38 CHF patients with various degrees of heart failure and in 21 healthy controls. Patients with CHF had increased plasma concentrations of tumor necrosis factor (TNF)alpha, interleukin-6, soluble TNF receptors and the soluble interleukin-6 receptor, glycoprotein (gp)130. They also had elevated ratios of TNFalpha/soluble TNF receptors and interleukin-6/soluble gp130 as well as enhanced interleukin-6 bioactivity in serum, suggesting inflammatory net effects. In addition to raised circulating levels of inflammatory cytokines, CHF patients with severe heart failure also had abnormalities in the levels of anti-inflammatory cytokines, with decreased levels of transforming growth factor beta1 and inadequately raised interleukin-10 in relation to the elevated TNFalpha concentrations. This dysbalance between inflammatory and anti-inflammatory cytokines was also found in monocyte supernatants from CHF patients. The abnormalities in the cytokine network were most pronounced in patients with the most severe heart failure, and several of the immunologic parameters, in particular soluble gp130, were correlated with variables reflecting deranged hemodynamic status. The present study analyzing the complexity of the cytokine network in CHF, demonstrates profound disturbances in the levels of both inflammatory and anti-inflammatory mediators with a marked dysbalance favoring inflammatory effects.