银屑病患者骨髓单一核细胞中端粒酶及其亚单位的表达

目的：观察银屑病患者骨髓单个核细胞（BMMNC）的端粒酶及其亚单位端粒酶逆转录酶（hTERT）mRNA的表达情况。方法：采用银染-端粒重复序列扩增（TRAP）法检测银屑病患者及正常人BMMNC端粒酶的表达,RT-PCR法检测患者及正常人hTERT mRNA的表达,同时以患者外周血单个核细胞（PBMC）作为自身对照。结果：30例患者骨髓中有4例呈端粒酶阳性,5例呈hTERT mRNA阳性,30例正常骨髓中有3例呈端粒酶阳性,6例呈hTERTmRNA阳性,患者外周血中未检测到端粒酶及hTERTmR-NA的表达。结论：骨髓中端粒酶的表达来源于造血细胞,银屑病患者和正常人骨髓中的端粒酶阳性率无显著差异,银屑病患者骨髓造血细胞增殖活性异常与端粒酶表达无关。     

HAX-1在SLE患者外周血单一核细胞中的表达及其亚细胞定位

目的 检测活动期SLE患者PBMC中抗凋亡基因HAX-1的表达水平，并进行亚细胞定位分析。方法 分别采用半定量PCR以及Western印迹试验从mRNA和蛋白水平上检测活动期SLE患者及正常人对照组PBMC中HAX-1的表达，并通过激光共聚焦显微镜观察HAX-1在SLE患者PBMC中的亚细胞定位。结果 活动期SLE患者PBMC中HAX-1在mRNA水平以及蛋白水平的表达均明显高于健康正常人（P值分别 = 0.000、0.007），且其相对表达水平与SLEDAI之间呈线性正相关；HAX-1在SLE患者PBMC中主要分布于线粒体内及内质网。结论 HAX-1可能通过抗凋亡机制抑制SLE患者体内淋巴细胞凋亡而导致其过度活化，从而参与SLE疾病发生发展过程。     

白介素22及其相关因子在银屑病患者血清及外周血单一核细胞中的表达

目的 探讨白介素（IL）-22及其相关因子IL-23p19、IL-6在寻常性银屑病患者外周血单一核细胞（PBMC）及血清中表达的意义。方法 采用实时定量RT-PCR法检测寻常性银屑病患者及正常人对照者外周血PBMC中IL-22 mRNA、IL-23p19 mRNA及IL-6 mRNA的水平;采用ELISA法检测寻常性银屑病患者及正常人对照者血清和PBMC培养上清中IL-22的分泌水平。结果 银屑病组和正常人对照组PBMC中IL-22 mRNA的相对表达量分别为4.48 ± 2.64和2.35 ± 0.91;IL-23p19 mRNA的相对表达量分别为6.07 ± 4.09和2.61 ± 1.46;IL-6 mRNA的相对表达量分别为3.87 ± 1.49和1.48 ± 0.62;差异均有统计学意义（P < 0.01）。ELISA检测发现,银屑病组和正常人对照组血清中IL-22的水平分别为（86.23 ± 25.58） ng/L和（43.67 ± 14.82） ng/L（P < 0.01）,PBMC培养上清中IL-22的水平分别为（119.11 ± 21.51） ng/L和（57.70 ± 13.17） ng/L（P < 0.01）。结论 寻常性银屑病患者PBMC和血清中过度表达IL-22,提示IL-22可能参与银屑病的发病机制。     

寻常型银屑病患者外周血环氧合酶同工酶的研究

目的 探讨进行期斑块状银屑病患者外周血单一核细胞（PBMC）中环氧合酶同工酶的表达及其在银屑病发病机制中的作用。方法 应用逆转录－聚合酶链反应（RT－PCR）检测了进行期斑块状银屑病患者及健康对照者各30例PBMC中构成性环氧合酶（COX－1）和诱导性环氧合酶（COX－2）mRNA的表达。结果 进行期斑块状银屑病患者PBMC中COX－2 mRNA的平均表达水平明显高于健康对照组（P＜0．01），且其mRNA的表达水平与其相应的面积与严重度积分（PASI）呈正相关（P＜0．01）；而两组间COX－1平均表达水平的差异无显著性（P＞0．05）。结论 进行期斑块状银屑病患者PBMC中COX－2 mRNA的表达增高，提示可能通过诱导前列腺素（PGs）的产生参与进行期斑块状银屑病的发病。     

银屑病患者外周血中 INS-R 基因外显子17基因多态性研究

目的：调查银屑病患者合并代谢性疾病及胰岛素抵抗发生率,探讨银屑病患者胰岛素抵抗与胰岛素受体基因外显子17基因多态性的关系。方法：在195例银屑病患者和210例健康对照组中,调查伴发肥胖、空腹血糖升高、高血脂等代谢性疾病的情况。应用聚合酶链反应—毛细管电泳方法检测66例胰岛素抵抗的银屑病患者和197例正常人外周血白细胞胰岛素受体基因外显子17多态性,分析其与胰岛素抵抗的相关性。结果：银屑病患者肥胖、高血脂、空腹血糖升高及胰岛素抵抗发生率明显高于对照组（P值均＜0．05）。胰岛素抵抗的银屑病患者和对照组胰岛素受体外显子17在密码子1058位点出现CAC-CAT突变,存在三种基因型C／C、C／T、T／T,但3种基因型频率在两组间差异无统计学意义（ P＞0.05）。结论：银屑病患者易发生代谢性疾病和胰岛素抵抗。外周血白细胞胰岛素受体基因外显子17基因多态性在银屑病患者胰岛素抵抗发生中可能不起作用。     

当归多糖对银屑病患者外周血单一核细胞NF-κB及IFN-γ的影响

目的：确定体外当归多糖对银屑病患者外周血单一核细胞（PBMC）与角质形成细胞（KC）NF-κB表达及IFN-γ分泌的影响.方法：将正常人KC＋银屑病患者PBMC混合培养（A组）,正常人KC和正常人PBMC混合培养作为作对照（B组）.当归多糖作用于两组,采用Western Blot检测混合细胞中NF-κB的蛋白表达水平,双抗体夹心ELISA法检测培养上清液中IFN-γ的水平.结果：A组混合培养体系中NF-κB p65蛋白表达水平及上清液中IFN-γ水平明显高于B组（P＜0.01）;经当归多糖作用后,A组混合培养体系中NF-κB p65蛋白表达水平及上清液中IFN-γ水平较处理前降低（P＜0.05）,而B培养体系中NF-κB p65蛋白表达水平及上清液中IFN-γ水平较处理前升高（P＜0.05）.结论：当归多糖可能通过抑制NF-κB活化、减少IFN-γ分泌,对银屑病治疗发挥作用.     

Notch1信号对银屑病患者外周血Th17细胞分化和功能的影响

目的 探讨Notch1信号对银屑病患者外周血Th17细胞分化和功能的影响。方法 取35例寻常性银屑病患者与32例健康对照外周血,流式细胞仪检测单个核细胞（PBMC）中Th17细胞占CD4+ T淋巴细胞的比例,实时荧光定量反转录PCR检测PBMC中维A酸相关孤儿核受体γt（RORγt）、白介素17（IL-17）、Notch1及发状分裂相关增强子1（Hes-1）mRNA表达水平,酶联免疫吸附试验检测血清及培养上清液中IL-17含量。分析Notch1 mRNA的表达与银屑病皮损面积和疾病严重程度评分（PASI）、Th17细胞比例、RORγt mRNA、IL-17 mRNA及蛋白表达水平的相关性。将银屑病患者PBMC分为0（对照组）、2.5、5.0、10.0、20.0 μmol/L γ分泌酶抑制剂（DAPT）处理组,检测DAPT阻断Notch1信号对PBMC中Th17细胞比例、RORγt及IL-17表达水平的影响。结果 银屑病患者PBMC中Th17细胞占CD4+ T细胞比例为2.863% ± 0.969%,RORγt、Notch1、Hes-1、IL-17 mRNA表达水平分别为5.255 ± 0.998、6.743 ± 1.756、6.384 ± 1.665、6.944 ± 1.626,IL-17血清含量为（36.444 ± 5.936） ng/L,均高于健康对照组［分别为0.604% ± 0.124%、1.530 ± 0.485、1.731 ± 0.456、1.627 ± 0.485、1.698 ± 0.329、（11.762 ± 2.260） ng/L,P < 0.01］。银屑病患者Notch1 mRNA表达水平与PASI、Th17细胞比例、RORγt mRNA表达水平、IL-17 mRNA表达水平及血清含量均呈正相关（r值分别为0.584、0.544、0.518、0.549、0.511,均P < 0.05）。5种不同浓度DAPT处理组银屑病患者PBMC中Th17细胞比例、RORγt mRNA表达水平、IL-17 mRNA表达水平及培养上清液含量差异有统计学意义（F值分别为79.527、82.239、78.086、80.558,均P < 0.01）,2.5、5.0、10.0、20.0 μmol/L DAPT组上述指标均低于对照组（均P < 0.05）,且随着DAPT作用浓度的增加,各指标呈降低趋势。结论 Notch1信号可促进银屑病患者外周血Th17细胞的分化及RORγt 、IL-17、Hes-1的表达。     

HO-1在银屑病患者皮损及外周血单一核细胞中的表达

目的：检测寻常型银屑病(PV)患者皮损及外周血单一核细胞(PBMC)中血红素氧合酶-1(HO-1)mRNA和蛋白的表达。方法：逆转录-聚合酶链反应(RT-PCR)法和免疫组化方法检测20例PV患者和10例正常人皮肤及PBMC中HO-1 mRNA 和蛋白的表达。结果： PV患者及正常对照组皮肤中HO-1mRNA表达相对含量分别为1.32±0.29和0.46±0.10，HO-1的免疫组化评分分别为10.25±2.36和2.65±1.03；患者及对照组PBMC中HO-1mRNA相对含量分别为0.25±0.14和0.22±0.16，HO-1染色阳性细胞的百分比为11.7％±3.44％和10.5％±4.06％，PV皮损中HO-1的mRNA和蛋白表达明显高于正常对照组(P<0.05)，而PV患者外周血PBMC中HO-1 mRNA和蛋白的表达水平与正常对照组差异无显著性(P>0.05)。结论： HO-1可能参与了银屑病皮损的发病过程。     

银屑病患者淋巴细胞与角质形成细胞体外培养NF-κB的表达

目的：探讨银屑病发病中淋巴细胞和角质形成细胞的相互作用及核因子调控机制。方法：从银屑病患者外周血分离单一核细胞（PBMC）．正常人皮肤体外培养获角质形成细胞（KC），两者混合培养，利用流式细胞仪分析培养体系及PBMC、KC各自的转录因子NF-κB表达，ELISA法测上清液中IL-8、ICAM—I含量。结果：银屑病患者培养体系及PBMC、KC中NF—κB和细胞因子的表达水平均高于健康对照组（P〈0．01）。结论：银屑病患者中NF—κB的活化增强是其发病的重要因素，同时提示抑制NF—κB活性的药物可能有助于银屑病的治疗。     

系统性红斑狼疮患者外周血单核细胞中IL—2及其受体mRNA的表达

目的：探讨系统性红斑狼疮（SLE）患者中白介素（IL－2）及其受体（IL－2R）表达的自然情况。方法：用逆转录－多聚酶链反应（RT－PCR）法检测17例SLE患者和10例正常人外周血单个核细胞（PBMC）中IL－2、IL－2RmRNA表达的水平。结果：与正常人相比，活动期SLE患者IL－2mRNA表达水平降低而IL－2mRNA表达水平增高（P＜0．01）。结论：SLE患者中IL－2R的表达未受IL－2的诱导。     

Lack of association of TNF-238A and -308A in Japanese patients with psoriasis vulgaris,psoriatic arthritis and generalized pustular psoriasis

Tumor necrosis factor (TNF)-alpha is one of the proinflammatory cytokines and immunomodulators, and has an important pathogenetic role in psoriasis. The TNF-alpha gene (TNFA) is in the human leukocyte antigen (HLA) class III locus on chromosome 6, which might be related to the pathogenesis of psoriasis. It has been suggested that some polymorphisms of the TNFA gene promoter, especially G to A conversions at nt-238 and -308 (TNF-238A and -308A), may be associated with psoriasis in Caucasians. We investigated single nucleotide polymorphisms (SNPs) of the TNFA gene promoter in Japanese psoriasis patients, including 18 with psoriasis vulgaris (PsV), 11 with psoriatic arthritis (PsA), two with generalized pustular psoriasis (GPP), and six with GPP with arthritis. The DNA fragment of the TNFA gene from nt-400 to -69 was amplified by polymerase chain reaction (PCR) and the products were sequenced. Although TNF-238A and other polymorphisms were not found in PsV and psoriatic arthritis patients, one male patient with GPP and PsA had TNF-308A. This suggests that TNFA gene promoter polymorphism in the region examined is less associated with the pathogenesis of psoriasis in Japanese patients, however there might be the possibility that TNFA gene promoter polymorphism is associated with GPP. Further investigation will be required to prove this.     

CTLA4Ig诱导银屑病Th1/Th2转换的体外研究

目的 研究银屑病的Th1/Th2反应模式,并观察CTLA4Ig在诱导银屑病Th1/Th2转换中的作用。方法 标本取自33例寻常型银屑病患者和20例正常人对照,利用植物血凝素(PHA)和葡萄球菌肠毒素B(SEB)单独及与CTLA4Ig协同刺激外周血单一核细胞(PBMC)增殖,通过酶联免疫吸附法检测PBMC培养上清液中白介素2(IL-2)、干扰素γ(IFN-γ)和白介素4(IL-4)水平。结果 SEB显着促进银屑病组3种细胞因子的分泌(P<0.01);进行期IFN-γ/IL-4的比值明显高于静止期(P<0.01).CTLA4Ig可以抑制IL-2、IFN-γ的分泌,并呈剂量依赖关系;而IL-4相对于IL-2及IFN-γ分泌是增强的;当CTLA4Ig浓度达到10μg/mL时,3种细胞因子的分泌均明显被抑制。结论 银屑病属于Th1型反应模式,CTLA4Ig可以下调Th1型细胞因子,上调Th2型细胞因子,提示CTLA4Ig具有诱导银屑病的Th1/Th2失衡向Th2转换的作用。     

CTLA-4 gene 49A/G polymorphism in Turkish patients with Behçet's disease

Genetic factors predisposing individuals to Behçet's disease (BD) are considered to play important roles in the development of the disease. Patients with BD exhibit elevated levels of pro‐inflammatory cytokines, and affected organs show a significant neutrophil and lymphocyte infiltration. Current evidence suggests that the activated lymphocytes contribute to neutrophil and endothelial cell activation in these patients. The cytotoxic T lymphocyte‐associated antigen (CTLA)‐4 molecule plays an important role in immune regulation by downregulating T‐cell activation, and the CTLA‐4 49A/G polymorphism in the exon 1 has been shown to be associated with a number of autoimmune diseases. In an attempt to demonstrate whether there is an association of the CTLA‐4 49A/G polymorphism with BD in the Turkish population, we genotyped 59 Turkish patients and 99 healthy individuals for single‐nucleotide polymorphisms. For this purpose, genomic DNA was obtained from the peripheral blood of individuals and the region of interest was amplified using PCR. Genotyping was performed using the BbvI restriction endonuclease. It was shown that the distribution of the CTLA‐4 exon 1 49A/G allele and genotype frequencies did not differ between patients with BD and healthy controls. However, allele and genotype frequencies of CTLA‐4 49 A and A/A were significantly higher in patients with ocular involvement compared with patients without these symptoms (90.6% vs. 65.1%, odds ratio (OR) = 9.67, P = 0.011; and 81.25% vs. 39.5%, OR = 9.56, P = 0.015, respectively). A statistically significant difference in the A allele frequency was observed in patients with erythema nodosum‐like lesions (86.1% vs. 65.8%, OR = 6.24, P = 0.04). There was also an increase in A/A genotype frequency, but the difference was not statistically significant (72.2% vs. 41.5%, OR = 6.5, P = 0.068). Our data suggest that BD patients with ocular involvement and erythema nodosum‐like lesions have a higher frequency of both the A allele and the A/A genotype at position 49 of exon 1 of CTLA‐4. These results may also indicate that CTLA‐4 is a disease‐modifying rather than a susceptibility gene for BD.     

Specific restriction fragment length polymorphism on the HLA-C region and susceptibility to psoriasis vulgaris

In psoriasis vulgaris, the HLA class I Cw6 specificity has previously been recognized as the most commonly associated antigen serologically. This study was carried out to investigate whether or not the gene controlling the susceptibility to psoriasis vulgaris existed on the HLA, especially the HLA-C region. At first, we analyzed the restriction fragment length polymorphism (RFLP) of 13 patients with psoriasis vulgaris and 6 healthy controls who were all positive for at least one allele of HLA-Cw6. To characterize RFLP in psoriasis patients who did not have HLA-Cw6, 12 patients and 10 healthy controls who had HLA-Cw7 were also examined. Southern hybridization of genomic DNA demonstrated that DNA polymorphisms of the HLA-C antigen gene could not be found in any psoriasis vulgaris patient whether HLA-Cw6 or Cw7. However, a 4.5 kb BamHI fragment and a 3.1 kb PstI fragment were lacking in some healthy controls who had either HLA-Cw6 or Cw7. This study suggests that the presence of RFLP in the HLA-C gene is associated with psoriasis vulgaris. These specific fragments may help predispose individuals to psoriasis vulgaris, or may be essential for the development of the disease.     

白癜风与CTLA-4基因外显子1 A/G~(49)多态性研究

目的探讨白癜风与细胞毒性T淋巴细胞相关抗原4(CTLA4)基因外显子1第49位点A/G多态性的相关性。方法采用多聚酶链反应限制性片段长度多态性(PCRRFLP)技术分析59例白癜风患者和124例健康对照者CTLA4基因外显子1第49位点的基因型。结果白癜风患者CTLA4基因外显子1第49位点的各基因型分布和等位基因频率与健康对照者相比,无显著性差异(χ2=0.735,P>0.05;χ2=0.684,P>0.05);当患者按有无自身免疫性疾病分类后,伴及不伴有自身免疫性疾病的白癜风患者CTLA4基因外显子1第49位点的各基因型分布及等位基因频率与健康对照者相比较无统计学意义(χ2=2.614,P>0.05;χ2=2.317,P>0.05);当患者按临床型别分类后,各型别等位基因频率与健康对照者相比无统计学意义(χ2=3.076,P>0.05)。结论CTLA4基因外显子1第49位碱基A/G多态性与白癜风的发病可能无相关性;将白癜风患者按有无自身免疫性疾病分类后,发现甚至与白癜风同时伴发的自身免疫性疾病也与CTLA4外显子1第49位点A/G49多态性无关。     

Abnormal histone modifications in PBMCs from patients with psoriasis vulgaris

Excessive keratinocyte proliferation is thought to be responsible for the formation and development of psoriasis vulgaris. Evidence indicates that epigenetic modifications are associated with aberrant gene expression, however, nothing is known about the status of histone modifications in psoriasis vulgaris. We investigated alterations in histone modifications in patients with psoriasis vulgaris. Global histone H3/H4 acetylation and H3K4/H3K27 methylation in peripheral blood mononuclear cells from 30 psoriatic patients and 20 healthy control subjects were quanti?ed by the EpiQuik(TM) global histone H3/H4 acetylation and H3K4/H3K27 methylation assay kit. The mRNA levels of 12 members of 3 classes of chromatin modifier genes were measured by real-time quantitative polymerase chain reaction. Compared with normal controls, global histone H4 hypoacetylation was observed in PBMCs from psoriasis vulgaris patients. There was a negative correlation between the degree of histone H4 acetylation and disease activity in patients as measured by PASI. Global levels of H3 acetylation, H3K4/H3K27 methylation did not significantly differ between psoriatic patients and controls. mRNA levels of P300, CBP and SIRT1 were significantly reduced in PBMCs from patients with psoriasis vulgaris compared with healthy controls, while mRNA expression levels of HDAC1, SUV39H1 and EZH2 was significantly increased in psoriatic patients.We conclude that histone modifications are aberrant in the PBMCs of psoriasis vulgaris patients.     

寻常性银屑病患者外周血单一核细胞IFN-γ受体和TNF-α受体mRNA表达的研究

目的 探讨寻常性银屑病患者外周血单一核细胞IFN-γ受体mRNA和TNF-α仅受体mRNA的表达及其在银屑病发病机制中的作用.方法 采集50例寻常性银屑病患者外周静脉血,分离单一核细胞后,以RT-PCR法检测其IFN-γ受体mRNA和TNF-α受体mRNA的表达,PASI评分法评估银屑病的严重程度.以24例正常人作对照.结果 50例银屑病患者外周血单一核细胞IFN-γ受体mRNA的表达均值为1.11±0.55,其中37例进行期患者为1.13±0.57,13例静止期患者为1.03±0.52,正常人对照组为0.72±0.17,银屑病患者显著高于正常人对照组(P=0.0034),进行期显著高于正常人对照组(P=0.008),而静止期与正常人对照组差异无统计学意义.正常人对照组和银屑病患者外周血单一核细胞TNF-α受体mRNA的表达水平差异无统计学意义.银屑病患者外周血单一核细胞IFN-γ受体mRNA表达水平与TNF-α受体mRNA表达水平及PASI值均无相关性.结论 IFN-γ受体mRNA在寻常性银屑病患者外周血单一核细胞中异常高表达,但与疾病的活动性无关.     

HCR基因单核苷酸多态性与维吾尔族银屑病的相关性

目的 探讨HCR基因与银屑病发病的相关性.方法 测定118例新疆维吾尔族寻常性银屑病及127例正常人对照组的HCR基因第4外显子第307位点基因多态性.从抗凝血中抽提DNA,用PCR-RFLP法和PCR产物直接测序法鉴定基因型.对结果进行统计学分析处理.结果 HCR-307位点核苷酸存在C、T二态性,表现为CC纯合、TF纯合、CT杂合三种基因型,病例组TF基因型频率明显高于对照组,差异有统计学意义(P<0.01).结论 HCR-307位点的C→T多态性与新疆维吾尔族寻常性银屑病具有相关性.     

The HCR gene on 6p21 is unlikely to be a psoriasis susceptibility gene

The PSORS1 locus in the human major histocompatibility complex on 6p21 has been consistently associated with psoriasis in populations of diverse ethnicity. The HLA-C allele Cw*0602, located therein, has been found in up to 67% of psoriasis patients but is no longer considered a candidate gene in itself. The alpha-helix coiled-coil rod homolog gene (HCR, previously Pg8) is located 110 kb from the HLA-C gene, positioned between the CDSN and SC1 genes, within a region thought to harbor a psoriasis gene (PSORS1). We investigated the HCR gene for disease association by direct sequencing of nine polymerase chain reaction products amplified from a series of Swedish psoriasis patients and controls. We found that HCR is a very polymorphic gene with 25 polymorphisms in the open reading frame alone, of which 10 demonstrated disease association; however, the relationship between HCR polymorphisms and HLA-Cw*0602 indicates that HCR cannot truly be considered a likely candidate gene. We investigated Cw*0602 association while stratifying for HCR single nucleotide polymorphisms. We also investigated HCR single nucleotide polymorphism association with the disease while stratifying for the presence of Cw*0602. We found that whichever single nucleotide polymorphism that was stratified for, there was still a strongly significant Cw*0602 association with psoriasis; however, when we stratified for Cw*0602 presence, only one silent polymorphism showed significant association. In a recent similar study this polymorphism was actually found to be decreased in psoriasis individuals. Thus we conclude that HCR polymorphisms display association with psoriasis due to linkage disequilibrium with Cw*0602 and is, therefore, unlikely to be directly involved in the development of psoriasis.