慢性特发性荨麻疹患者嗜碱粒细胞差异表达基因的研究

目的: 检测慢性特发性荨麻疹(CIU)患者外周血嗜碱粒细胞基因表达谱。方法: 分离提取2例CIU患者和2名健康人外周血嗜碱粒细胞,提取总RNA;利用cRNA标记方法对样品进行荧光标记,然后用于芯片杂交;用LuxScan 10K/A双通道激光扫描仪进行芯片扫描;采用LuxScan 3.0图像分析软件对芯片图像进行分析;然后对芯片上的数据用Lowess方法进行归一化;最后以差异为2倍的标准来确定差异表达基因。结果: CIU患者嗜碱粒细胞与健康对照组相比存在差异表达基因共计30个。其中表达上调基因12个,表达下调基因8个,功能不明基因10个。结论:CIU患者和正常人嗜碱粒细胞的一些基因表达差异可能与CIU发病有关。     

孕酮在淋球菌引起的中性粒细胞炎症反应中的调节作用

目的 研究孕酮在淋球菌引起的中性粒细胞炎症反应中的作用。方法 提取正常人外周血中性粒细胞,根据是否加入孕酮,将其分为孕酮组、淋球菌组、干预组（孕酮 + 淋球菌）及对照组。荧光定量RT-PCR分别测定在0、3、8、12 h各组中性粒细胞中诱导型一氧化氮合酶（iNOS）、TNF-α、IL-1β mRNA含量,并用Western印迹测定iNOS蛋白水平。结果 淋球菌组和干预组中iNOS、TNF-α、IL-1β mRNA表达水平均升高;淋球菌组在8 h达到高峰,以后逐渐下降;干预组三者水平明显低于淋球菌组（P < 0.05）。而孕酮组、对照组各因子含量无明显变化。Western印迹结果显示,淋球菌组和干预组iNOS蛋白表达水平亦升高,前者明显高于后者（P < 0.05）。结论 孕酮下调中性粒细胞iNOS、TNF-α及IL-1β的表达,抑制淋球菌引起的中性粒细胞炎症反应,这一机制可能在女性无症状感染中起作用。     

慢性自发性荨麻疹患者外周血嗜碱粒细胞CD63和CD203c的表达

目的：检测慢性自发性荨麻疹患者中外周血嗜碱粒细胞的活化状态。方法：采用流式细胞技术分别检测慢性自发性荨麻疹患者风团不同持续时间下（A组<2 h,15例;B组12~24 h,15例）、治疗后患者（C组,15例）及健康对照组（D组,15例）外周血嗜碱粒细胞CD63+和CD203c+的表达情况。结果：A、B、C三组外周血嗜碱粒细胞CD63+和CD203c+活化百分率（0.097±0.019,0.072±0.015,0.051±0.012）均高于对照D组（0.007±0.002,P<0.05）。A、B、C三组之间两两比较无显著差异（P>0.05）。结论：活化的嗜碱粒细胞可能参与了慢性自发性荨麻疹的发病过程,但与风团的持续时间无相关性。     

转录因子Aiolos基因在SLE患者外周血单一核细胞中的表达

目的 探讨SLE患者外周血单一核细胞（PBMC）中转录因子Aiolos的表达水平。方法 采用Western印迹法分别检测19例SLE活动期患者及13例SLE稳定期患者PBMC中转录因子Aiolos蛋白的表达水平,并以30例健康志愿者作对照。对SLE患者Aiolos蛋白表达水平与SLE疾病活动指数（SLEDAI）进行相关分析。结果 PBMC中转录因子Aiolos蛋白表达相对值正常对照组为0.81 ± 0.09,SLE组为0.56 ± 0.17,两组比较差异有统计学意义（P < 0.01）;SLE活动期组（0.52 ± 0.14）与稳定期组（0.65 ± 0.19）比较,差异有统计学意义（P < 0.05）;SLE患者转录因子Aiolos蛋白的表达与SLEDAI呈显著负相关（r = -0.65,P < 0.01）。结论 SLE患者PBMC中转录因子Aiolos的表达水平显著降低,且与SLE疾病活动指数呈一定相关性。     

慢性特发性荨麻疹患者外周血单核细胞CCR3和CX3CR1 mRNA的表达

目的探讨趋化因子受体CCR3和CX3CR1在慢性特发性荨麻疹(CIU)患者外周血单核细胞(PBMC)中的表达及其与疾病的相关性。方法确诊为CIU的患者45例和正常人对照30例,收集其PBMC,提取RNA。应用反转录-聚合酶链反应(RT-PCR)法检测入选者的CCR3和CX3CR1 mRNA表达水平。结果 CCR3 mRNA表达水平比较,CIU患者组(0.69±0.22)高于正常对照组(0.51±0.26),差异有统计学意义(P<0.01)。而CX3CR1 mRNA表达水平比较,CIU患者组(0.55±0.27)与正常对照组(0.53±0.14)的差异无统计学意义(P>0.05)。结论 CIU患者CCR3 mRNA表达水平比正常对照组增高,而CX3CR1 mRNA表达水平与正常对照组差异不显著。提示CCR3可能参与CIU的发生机制。     

白塞综合征患者外周血中性粒细胞FcγRⅡA mRNA的表达及其意义

目的 探讨白塞综合征患者外周血中性粒细胞FcγRⅡA mRNA表达及其与血浆髓过氧化物酶（MPO）活性的关系。方法 采集25例白塞综合征患者活动期及其中15例患者经治疗后的非活动期静脉血,分离中性粒细胞后,以RT-PCR检测其FcγRⅡA mRNA的表达。采用分光光度法测定血浆MPO活性（代表中性粒细胞激活）。以20例正常人作对照。结果 白塞综合征患者外周血中性粒细胞FcγRⅡA mRNA的表达在活动期为1.801 ± 0.829,非活动期为0.820 ± 0.625,血浆MPO活性分别为32 ± 5 U/L和27 ± 4 U/L,两者表达在活动期组均高于非活动期组（P ＜ 0.01）,非活动期组与正常人对照组比较,差异均无统计学意义（P ＞ 0.05）。白塞综合征患者外周血中性粒细胞FcγRⅡA mRNA的表达水平与血浆MPO活性呈正相关（r = 0.39,P ＜ 0.01）。结论 白塞综合征活动期外周血中性粒细胞FcγRⅡA mRNA表达升高。推测中性粒细胞的FcγRⅡA可能与白塞综合征中性粒细胞的激活有关。     

瘦素调节HaCaT细胞角蛋白17的表达

目的 探讨瘦素对HaCaT细胞角蛋白17（K17）表达的影响。 方法 体外培养HaCaT细胞,给予100 ng/ml的瘦素作用24 h,应用实时PCR检测K17 mRNA表达水平、Western印迹及免疫荧光染色法检测K17蛋白表达水平变化。 结果 与阴性对照组（1.000 0 ± 0.000 0）相比较,瘦素组（3.086 7 ± 0.186 1）K17 mRNA表达显著升高,差异有统计学意义（P < 0.01）。Western印迹结果表明,瘦素组K17蛋白较阴性对照组显著上调,细胞免疫荧光染色结果与RT-PCR、Western印迹结果相符。与单纯使用瘦素组（2.242 7±0.188 7）相比较,STAT3抑制剂组和Erk1/2抑制剂组K17 mRNA分别为0.674 1 ± 0.060 0、0.855 0 ± 0.390 3,Western印迹和细胞免疫荧光染色显示,两个抑制剂组的K17蛋白较瘦素组均显著下调,差异均有统计学意义（P < 0.01）。 结论 瘦素可以诱导HaCaT细胞表达K17,其机制可能与激活STAT3、Erk1/2信号转导途径有关。     

荨麻疹

20110560慢性特发性荨麻疹患者嗜碱粒细胞差异表达基因的研究/马一平(中国医科院皮研所),姚煦,林麟…∥中国麻风皮肤病杂志.-2010,26(11).-756~758分离提取2例慢性特发性荨麻疹(CIU)患者和2名健康人外周血嗜碱粒细胞,提取总RNA;用cRNA标记方法对样品进行荧光标记,用于芯片杂交;用Luxs-can10K/A双通道激光扫描仪进行芯片扫描;采用Lux-     

培养硬皮病皮损成纤维细胞Smad3 DNA结合活性的研究

目的：探讨培养硬皮病成纤维细胞中磷酸化转录因子Smad3的表达和转录因子Smad3的DNA结合活性和表达量。方法：4例硬皮病患者和4例正常人对照进入本研究。采用Western Blot检测了磷酸化Smad3在培养硬皮病成纤维细胞全细胞裂解液中的表达;采用电泳迁移率实验（electrophoretic mobility shift assay,EMSA）检测了培养硬皮病成纤维细胞在核蛋白中Smad3的DNA结合活性,并对其进行了密度扫描和定量分析。结果：磷酸化转录因子Smad3的表达在培养硬皮病成纤维细胞全细胞裂解液中呈高表达,所有正常人对照为阴性。在培养硬皮病成纤维细胞核蛋白中,转录因子Smad3的DNA结合活性是显著增高的;经密度扫描,是正常人对照的21倍;培养正常人成纤维细胞核蛋白中转录因子Smad3的DNA结合活性仅略高于本底颜色。结论：磷酸化转录因子Smad3高表达和Smad3的DNA结合活性显著增高,提示转录因子Smad3在硬皮病纤维化的病理发生过程中具有重要作用。     

氮杂胞苷对SLE患者外周血T细胞Th2细胞因子基因表达的影响

目的探讨DNA甲基化抑制剂氮杂胞苷对SLE患者和正常人外周血T细胞DNA甲基转移酶1和细胞因子IL-4，IL-6，IL-10表达的影响。方法分离活动期SLE患者（n=15）、非活动期SLE患者（n=13）以及正常人对照（n=14）外周血T细胞，经PHA刺激1天后，分为甲基化抑制组和非抑制组进行培养，分别加入或不加氮杂胞苷继续培养3天；用RT—PCR方法测定各组外周血T细胞IL-4，IL-6，IL-10和DNA甲基转移酶1（Dnmt1）mRNA的表达水平。结果①非抑制组内，活动期、非活动期SLE患者的IL-4，IL-6，IL-10mRNA表达均高于正常人对照，Dnmt1mRNA表达显著下降，差异有统计学意义（P值均〈0．05）；并且，活动期、非活动期SLE患者DNA甲基转移酶1的表达与IL-4，IL-6，IL-10mRNA水平呈负相关（P值均〈0．05）。②与非抑制组比较，甲基化抑制组中的正常人T细胞IL-4，IL-6，IL-10mRNA表达增加，而Dnmt1表达则明显降低，差异均有统计学意义（P值均〈0．05）。甲基化抑制组中的活动期、非活动期SLE患者与非抑制组比较，T细胞表达Th2细胞因子、DNA甲基转移酶1表达差异无统计学意义（P值均〉0．05）。结论T细胞表达Th2细胞因子与基因组的DNA甲基化程度相关。     

Activated status of basophils in chronic urticaria leads to interleukin-3 hyper-responsiveness and enhancement of histamine release induced by anti-IgE stimulus

BACKGROUND: Basophils and mast cells are the main target cells in chronic idiopathic urticaria (CIU). Besides the basopenia, intrinsic defects of the anti-IgE cross-linking signalling pathway of basophils have been described in CIU. OBJECTIVES: We sought to investigate the profile of expression of activation markers on basophils of patients with CIU and to explore the effect of interleukin (IL)-3 priming upon anti-IgE cross-linking stimuli through expression of activation markers and basophil histamine releasability. METHODS: Evaluation of the surface expression of FcepsilonRIalpha, CD63, CD203c and CD123 on whole blood basophils of patients with CIU undergoing autologous serum skin test (ASST) was performed by flow cytometry. The effect of pretreatment with IL-3 in the anti-IgE response was analysed by the expression of basophil activation markers and histamine release using enzyme-linked immunosorbent assay. RESULTS: Blood basophils of patients with CIU were reduced in number and displayed increased surface expression of FcepsilonRIalpha, which was positively correlated with the IgE serum levels. Upregulation of expression of both surface markers CD203c and CD63 was verified on basophils of patients with CIU, regardless of ASST response. High expression of IL-3 receptor on basophils was detected only in ASST+ patients with CIU. Pretreatment with IL-3 upregulated CD203c expression concomitantly with the excreting function of blood basophils and induced a quick hyper-responsiveness to anti-IgE cross-linking on basophils of patients with CIU compared with healthy controls. CONCLUSIONS: Basophils of patients with CIU showed an activated profile, possibly due to an in vivo priming. Functionally, basophils have high responsiveness to IL-3 stimulation, thereby suggesting that defects in the signal transduction pathway after IgE cross-linking stimuli are recoverable in subjects with chronic urticaria.     

激活蛋白-1在皮肌炎患者外周血单一核细胞中的表达及糖皮质激素对其表达的影响

目的 探讨激活蛋白-1(AP-1)在皮肌炎患者外周血单一核细胞(PBMC)中的表达及糖皮质激素对其表达的影响.方法 分别提取15例正常人和20例皮肌炎患者(为未使用过糖皮质激素的初发患者或停止糖皮质激素治疗1个月以上者,初发12例,复发8例)的PBMC,每例的PBMC等分为2份,一份加入含80 μmol/L地塞米松和10％小牛血清的RPMI 1640培养液培养48 h后待用,另一份直接-80℃保存待用.用电泳迁移率改变实验检测不同组别PBMC中AP-1的活性.结果 AP-1在正常人PBMC中为低表达(灰度面积值为4.93±0.15 mm2).皮肌炎初发组和复发组患者PBMC加地塞米松前的AP-1表达(灰度面积值分别为30.23±0.49 mm2和34.79±0.61 mm2)均明显高于地塞米松处理后AP-1表达(灰度面积值分别为5.59±0.39 mm2和5.69±0.39 mm2),地塞米松处理前复发组PBMC的AP-1活性高于初发组(P＜0.01).结论 AP-1活性增强可能是导致皮肌炎炎症反应及复发的重要因素之一.糖皮质激素可以一定程度抑制AP-1活性.     

The effect of LXR activators on AP-1 proteins in keratinocytes

Oxysterols, via activation of liver X receptor (LXR), regulate keratinocyte differentiation by stimulating transglutaminase cross-linking of several constituent proteins leading to the formation of the cornified envelope. We previously reported that oxysterols increase the expression of one of these cross-linked proteins, involucrin, and that this effect can be abolished by mutations of the distal activator protein (AP)-1 response element in the involucrin promoter. Furthermore, oxysterols increase AP-1 binding in an electrophoretic gel mobility shift assay and increase the expression of an AP-1 reporter. In this study, we describe the individual components of the AP-1 complex that are involved in the oxysterol-mediated AP-1 activation and stimulation of keratinocyte differentiation. We identified Fra-1 within the AP-1 DNA binding complex by supershift analysis of nuclear extracts from oxysterol-treated, cultured keratinocytes and confirmed that oxysterol treatment increased the levels of Fra-1 by western blot analysis. Additionally, on Western and Northern analysis, oxysterol treatment increased two other AP-1 proteins, Jun-D and c-Fos, whereas Fra-2, Jun-B, and c-Jun were not changed. Similar alterations in AP-1 proteins occurred when 25-OH-cholesterol or non-steroidal LXR agonists (GW3965, TO-901317) were used. These results indicate that oxysterols induce specific AP-1 proteins, thereby activating involucrin, one of the genes required for epidermal differentiation.     

The effect of L-thyroxine treatment on chronic idiopathic urticaria and autoimmune thyroiditis

Autoimmune thyroiditis (AT) is more prevalent in patients with chronic idiopathic urticaria CIU) than in the general population. Previous small studies without any controlled comparison reported that CIU remits in patients with CIU and AT treated with L-thyroxine. To determine whether l-thyroxine treatment can improve the clinical course of CIU in patients with the co-occurrence of AT and CIU. A total of 749 patients with CIU were retrospectively studied. Clinical and laboratory evaluation and classification of chronic urticaria were performed according to the EAACI/GA(2)LEN/EDF/WAO guidelines. After L-thyroxine treatment for 53 ± 19 days, euthyroidism was restored in all subjects. Urticaria activity score (UAS) was evaluated at baseline and after three and six months. The control group consisted of matched 44 euthyroid subjects with CIU. A total of 44 (5.9%) patients were diagnosed to have hypothyroidism related to AT. Autologous serum skin test (ASST) was found to be positive in 17 (38.6%) of them. There was no statistically significant difference in baseline UAS, between the ASST+ (3.94 ± 1.52) and the ASST- (3.63 ± 1.42; P = 0.27) hypothyroid subjects and the euthyroid CIU controls (3.73 ± 1.74). During the L-thyroxine treatment, a significant reduction of UAS was observed in both hypothyroid ASST+ and ASST- subjects. However, the mean UAS after three and six months of L-thyroxine treatment remained not significantly different from that in control euthyroid subjects with CIU. L-Thyroxine treatment has no effect on the course of CIU in patients with CIU and AT.     

慢性特发性荨麻疹患者外周血单个核细胞分泌IFN-γ和IL-4水平检测及意义

目的　探讨Th1 /Th2细胞因子水平变化在慢性特发性荨麻疹(CIU)发病机制中的作用。方法　采用ELISA法检测37例CIU患者外周血单个核细胞(PBMC)培养上清中的细胞因子IFN γ和IL 4。结果　CIU患者IFN γ水平低于正常对照组(P<0. 05),其浓度分别为170. 58±230. 28pg/ml及340. 76±220. 35pg/ml;CIU患者IL 4水平高于正常对照组(P<0. 05),其浓度分别为41. 53±12. 57pg/ml与27. 01±13. 54pg/ml。结论　Th2细胞功能亢进及Th1功能低下在CIU发生发展中可能发挥一定的作用。     

Human leucocyte antigen class II associations in chronic idiopathic urticaria

The major histocompatibility complex (MHC) acts as a marker for self during T-cell ontogeny and is associated with the pathogenesis of many autoimmune diseases. Recent investigations have shown about 30% of patients with chronic idiopathic urticaria (CIU) have IgG autoantibodies against the high-affinity IgE receptor, FcepsilonRI, or IgE. A link between MHC class II alleles and CIU has not been reported previously. DNA was extracted from blood of 100 Caucasian patients with CIU, and the MHC class II type determined using the polymerase chain reaction with sequence-specific primers, testing for DRB and DQB1 alleles. The frequency of alleles in CIU patients was compared with that found in 603 controls. Further human leucocyte antigen (HLA) typing on patient subsets, classified by the patients' responses to intradermal injection of autologous serum and their serum-induced histamine release from basophil leucocytes of healthy donors, was undertaken. HLA DRB1*04 (DR4) and its associated allele, DQB1*0302 (DQ8), are raised in CIU patients compared with a control population (P = 2 x 10-5 and P = 2 x 10-4, respectively). HLA DRB1*15 (DR15) and its associated allele, DQB1*06 (DQ6), are significantly less frequently associated with CIU. The HLA DRB1*04 association is particularly strong (corrected P = 3.6 x 10-6) for patients whose serum has in vivo and in vitro histamine-releasing activity. HLA class II typing is consistent with the concept that CIU is a heterogeneous disease, and supports an autoimmune pathogenesis in a subset of patients.     

慢性特发性荨麻疹相关抗体与发病的关系

【摘要】 目的 探讨抗高亲和力IgE受体（FcεRI）抗体、抗IgE抗体、抗幽门螺杆菌（HP）抗体和抗甲状腺球蛋白抗体（TGAb）与慢性特发性荨麻疹（CIU）发病的关系。 方法 设（CIU）组、急性荨麻疹（AU）组和健康对照组,每组100例受试者。每例受试者均进行自体血清皮肤试验,荧光酶联免疫吸附法检测过敏原,血清总IgE、抗FcεRI抗体、抗IgE抗体、抗HP抗体、TGAb水平及阳性率,将CIU组与AU组和健康对照组比较。结果 CIU组自体血清皮肤试验阳性率为53%,AU组为12%,健康对照组未发现阳性者。CIU组和健康对照组过敏原检测均为阴性,而AU组能检测到食物或吸入物过敏原,阳性率为86%。 CIU组抗FcεRI抗体和抗IgE抗体水平均高于AU组和健康对照组（P ＜ 0.05）; CIU患者IgE水平低于健康对照组（T = 190.00,P ＜ 0.05）,而AU组IgE水平高于健康对照组（T = 226.00,P ＜ 0.05）; 自体血清皮肤试验阳性CIU患者抗FcεRI抗体水平较阴性患者高（T = 101.73,P ＜ 0.05）,抗IgE抗体在自体血清皮肤试验阳性和阴性CIU患者间,差异无统计学意义（T = 312.04,P ＞ 0.05）; CIU组、AU组、和健康对照组抗HP抗体阳性率分别为29%、19%和23%,TGAb阳性率分别为18%、15%和11%,抗HP抗体和TGAb阳性率在三组间比较,差异均无统计学意义（P ＞ 0.05）; 抗HP抗体阳性的CIU患者中,抗FcεRI抗体阳性率较AU患者和健康人高（P ＜ 0.01）;抗IgE抗体阳性率与AU患者和健康人差异无统计学意义（P ＞ 0.05）。TGAb阳性的CIU患者中,抗FcεRI抗体阳性率较AU患者和健康人高（P ＜ 0.01）,抗IgE抗体阳性率与AU患者和健康人比较,差异无统计学意义（P ＞ 0.05）。结论 CIU存在抗FcεRI和抗IgE自身抗体,可能在自身免疫性荨麻疹发病中起一定作用。 【关键词】 荨麻疹; 自身抗体; 免疫球蛋白E; 皮肤试验     

Chronic idiopathic urticaria: comparison of the clinical features of patients with and without anti-FcepsilonRI or anti-IgE autoantibodies

BACKGROUND: Previous studies defining the clinical features of patients with chronic idiopathic urticaria (CIU) were performed before the identification of functional autoantibodies against FcepsilonRI and/or IgE, now known to be present in approximately 30% of patients with CIU. OBJECTIVE: Our purpose was to determine whether there are differences between patients with and those without autoantibodies in the clinical features or severity of CIU. METHODS: The clinical features of 107 patients with CIU were evaluated prospectively. Patients were identified as having functional autoantibodies on the basis of the serum-evoked histamine release in vitro from the basophils of 2 healthy donors. RESULTS: Patients with autoantibodies (31%) had more wheals (P = .005), a wider distribution of wheals (P = .009), higher itch scores for the most severe episodes of itching (P = .002), more systemic symptoms (P = .03), and lower serum IgE levels (P < .0005) than patients without autoantibodies. CONCLUSION: The presence of autoantibodies indicates a subset of patients with more severe CIU.