尖锐湿疣组织中微卫星DNA的改变与HPV感染的相关性

目的探讨特定位点的微卫星不稳定性(MSI)及杂合性丢失(LOH)在尖锐湿疣(CA)组织中的检出情况及其与人乳头瘤病毒(HPV)感染的关系。方法选取D3S1234,D3S1611,D9S171,D10S1687和D17S16785个微卫星位点,应用聚合酶链反应(PCR)和变性聚丙烯酰胺凝胶电泳-银染色技术对40例CA组织及11例CA癌变组织进行微卫星分析,并采用核酸分子快速导流杂交基因芯片技术检测HPV的基因分型。结果 CA及CA癌变组织中LOH的总检出率分别为22.50%(9/40)和63.64%(7/11),差异有统计学意义(P<0.05),两组中LOH发生频率最高的位点均为D9S171,其次为D17S1678位点。MSI的发生率较低,CA组中频率为10.00%(4/40),CA癌变组为18.18%(2/11)。CA组及CA癌变组标本,高危型HPV感染者LOH阳性率明显高于低危型HPV感染者,差异有统计学意义(P=0.019);两组中MSI阳性的6例样本均为高危型HPV感染。结论高频LOH位点D9S171和D17S1678连锁的抑癌基因p16,p53可能在CA癌变的发生过程中起着重要作用;另外,该结果也说明HPV感染,尤其高危基因型病毒是CA癌变的因素之一。微卫星DNA的改变与高危型HPV的感染有一定的相关性。     

转录因子CCAAT/增强子结合蛋白α在银屑病皮损中的表达及意义

目的 探讨转录因子CCAAT/增强子结合蛋白α（C/EBP-α）在寻常性银屑病皮损中的表达及与角质形成细胞异常增殖及皮损严重程度间的相关性。 方法 用免疫组化二步法及Western印迹检测30例寻常性银屑病患者皮损和30例健康对照皮肤的表皮中C/EBP-α。免疫组化法检测Ki-67的表达,并根据Ki-67阳性表达数量计算增殖指数,Pearson相关分析法分析寻常性银屑病皮损中C/EBP-α的表达水平与角质形成细胞Ki-67增殖指数及银屑病皮损面积和严重程度指数（PASI评分）间的相关性。 结果 C/EBP-α主要在角质形成细胞胞质中表达,寻常性银屑病皮损中C/EBP-α的表达较健康对照皮肤明显下调（t = 7.82,P ＜ 0.05）。Ki-67主要在角质形成细胞胞核中表达,寻常性银屑病皮损中角质形成细胞的增殖指数明显高于健康对照皮肤（t = 4.54,P ＜ 0.05）。经Pearson相关分析,寻常性银屑病皮损中C/EBP-α表达水平与增殖指数呈负相关,与PASI评分呈负相关。Western印迹结果亦显示,C/EBP-α在银屑病皮损处的表达量显著下调。 结论 C/EBP-α在寻常性银屑病皮损中表达下调,可能参与了寻常性银屑病的发病过程。     

一氧化氮对角质形成细胞cGMP含量和白介素8表达的影响

近年研究表明银屑病皮损角质形成细胞、内皮细胞诱导型一氧化氮合酶(iNOS)的表达显着增加,银屑病皮损区一氧化氮(NO)的产生远高于非皮损区,非皮损区又显着高于正常人皮肤^[1-3]。我们观察了NO对体外培养角质形成细胞cGMP含量、白介素-8(IL-8)表达的影响。     

寻常性银屑病皮损表皮中Smad7表达的检测及其临床意义

目的:探讨Smad7基因及其蛋白在寻常性银屑病皮损区表皮中的表达及其意义。方法:采用逆转录(RT)-PCR和SP免疫组化法分别检测寻常性银屑病皮损和正常对照皮肤中Smad7的表达。结果:寻常性银屑病皮损中Smad7表达水平上调。与正常对照皮肤相比,寻常性银屑病皮损区表皮角质形成细胞Smad7的免疫组化染色显著增强(P<0.01)。结论:寻常性银屑病皮损区表皮Smad7的过度表达可能是通过阻断转化生长因子(TGF)-β信号转导,从而有助于银屑病皮损区表皮过度增生。     

寻常型银屑病表皮中固醇调节元件结合蛋白-1表达模式的研究

目的:评价固醇调节元件结合蛋白-1(SREBP-1)在寻常型银屑病表皮角质形成细胞中的表达模式和意义.方法:采用免疫组化方法检测SREBP-1在5例正常人表皮、17例寻常型银屑病皮损和非皮损表皮中的表达,并用图像分析方法进行半定量分析.结果:SREBP-1在正常人表皮角质形成细胞中呈细胞核表达;在银屑病非皮损及皮损表皮细胞中出现入核受阻;而SREBP-1在银屑病皮损中的表达强度较非皮损下降(P=0.047).结论:银屑病表皮细胞中SREBP-1入核受阻,及其在皮损中表达强度的下调,可能参与银屑病的发病过程.     

银屑病角质形成细胞p14ARF基因甲基化的研究

目的:检测银屑病皮损角质形成细胞p14ARF基因启动子区甲基化,探讨其与银屑病发病的关系。方法:利用甲基化特异性PCR技术检测37例银屑病及35例正常人角质形成细胞p14ARF基因启动子区甲基化状态。结果:37例银屑病皮损角质形成细胞p14ARF基因启动子区甲基化阳性率为43.2%(16/37),显著高于正常对照(χ2=13.5,P<0.05)。结论:p14ARF基因启动子区甲基化与银屑病角质形成细胞过度增殖具有一定的关系。     

PTEN、Akt在寻常型银屑病皮损中的表达及意义

目的:检测PTEN、Akt蛋白在寻常型银屑病皮损中的定位及表达水平,探讨其在银屑病角质形成细胞异常增殖中的作用。方法:收集30例寻常型银屑病患者的皮损及30例健康对照皮肤组织,采用免疫组化及Western blot法检测PTEN、Akt的表达,比较两组之间PTEN、Akt的表达水平。结果:免疫组化结果显示,PTEN、Akt蛋白均主要表达于表皮角质形成细胞的胞浆中,其中PTEN在寻常型银屑病皮损的角质形成细胞中为淡黄色颗粒,在健康对照皮肤角质形成细胞中为深黄色颗粒;Akt在寻常型银屑病皮损的角质形成细胞中为棕黄色颗粒,在健康对照皮肤角质形成细胞中为浅黄色颗粒。Western blot结果显示,与健康对照皮肤比较,寻常型银屑病皮损中PTEN蛋白的表达明显下调[(0.78±0.21)vs(1.46±0.34),t=9.39,P0.05],Akt蛋白的表达水平明显上调[(1.83±0.28)vs(0.92±0.16),t=15.56,P0.05]。结论:PTEN的低表达、Akt的高表达可能促进了银屑病皮损中角质形成细胞的异常增殖。     

寻常性银屑病患者HLAⅠ类分子A、B、C位点甲基化的研究

目的探讨寻常性银屑病患者表皮中HLA Ⅰ类分子重链A、B、C位点启动子区甲基化与疾病的关系。方法 甲基化特异性PCR技术检测46例寻常性银屑病患者皮损和非皮损表皮中的HLAⅠ类分子重链A、B、C位点启动子区域CpG岛的甲基化率,与28例正常人皮肤组织进行对照研究。PASI评分评价银屑病患者皮损的严重程度。结果 在银屑病患者皮损、非皮损和正常人皮肤组织中,重链A位点均未检测到发生甲基化,B位点的甲基化率分别是4.35％( 2/46)、4.35％( 2/46)和0,C位点的甲基化率分别是4.35％ (2/46)、21.74％( 10/46)和0。3组样本比较,C位点启动子区甲基化率非皮损明显高于皮损和正常人皮肤组织,但与病情严重程度无关;HLA Ⅰ类分子重链A和B位点差异均无统计学意义。结论 寻常性银屑病患者HLA Ⅰ类分子C位点启动子区甲基化异常。     

寻常型银屑病患者17号染色体上两个等位基因频率的检测

目的初步探讨2个微卫星标记D17S784及D17S928与寻常型银屑病的相关性。方法用多重PCR和微卫星荧光标记-半自动基因扫描法以及GenotypeTM软件进行基因分型。结果银屑病患者与D17S784、D17S928具有显著相关性(P<0.001)，这两个微卫星位点的等位基因片段频率分布在银屑病组与健康对照组中差异具有显著性。结论D17S784与D17S928这2个连续标记可能是寻常型银屑病易感性的候选区域之一。     

Gadd45a基因在寻常型银屑病皮损中的表达及意义

目的:检测寻常型银屑病皮损中生长阻滞和DNA损伤基因(Gadd45a)mRNA的表达水平及Gadd45a蛋白在皮损中的表达部位,探讨其在寻常型银屑病角质形成细胞异常增殖中的作用。方法:收集20例寻常型银屑病患者的皮损及20例健康对照皮肤组织,RT-PCR法检测Gadd45a mRNA的表达水平,采用免疫组化法检测Gadd45a蛋白的表达部位,并进行比较。结果:RT-PCR结果显示,与健康对照皮肤比较,寻常型银屑病皮损中Gadd45a mRNA的相对表达量为4.51±1.57,差异有统计学意义(P0.05);免疫组化结果显示Gadd45a蛋白主要表达于表皮角质形成细胞的胞浆中,在寻常型银屑病病皮损的角质形成细胞中为棕黄色颗粒,在健康对照皮肤角质形成细胞中为阴性或者偶见。结论:Gadd45a的高表达可能在寻常型银屑病皮损角质形成细胞的良性异常增殖中发挥了重要作用。     

神经生长因子受体P75和P140trkAmRNA在寻常型银屑病患者皮损中的表达

目的研究神经生长因子(nerve growth factor,NGF)受体P75和P140trkA在寻常型银屑病发病机制中的作用.方法应用原位杂交技术检测了33例寻常型银屑病患者(18例为进展期,15例为静止期)和10例正常人皮肤组织中两受体(P75和P140trkA)mRNA的表达情况.结果与正常人皮肤比较,寻常型银屑病皮损及非皮损中P75和P140trkA受体mRNA的表达明显上调,且皮损中的表达明显高于非皮损区(P＜0.01);进展期患者皮损与非皮损中P75和P140trkA受体mRNA的表达分别高于静止期患者的皮损与非皮损区(P＜0.01).结论神经生长因子及其两受体可能是参与银屑病病理机制的重要因子.     

Cyclosporin A induces the unfolded protein response in keratinocytes

Psoriasis vulgaris is a chronic inflammatory disorder of the skin, in which activation of keratinocytes and crosstalk between keratinocytes and T cells or dendritic cells are considered to be involved in the pathogenesis of psoriasis vulgaris. Cyclosporin (Cy) A, an immunomodulator, has been used for the treatment of psoriasis vulgaris, but the mechanism of its action on keratinocytes has not been well elucidated as its function on T cells is well known. Previous study indicated that the expression of the unfolded protein response (UPR) markers, GRP78/Bip and HRD1 were poorly expressed in psoriasis vulgaris. To investigate if the UPR in keratinocytes is involved in the pathogenesis of psoriasis vulgaris we assessed immunocytochemistry of normal human skin and psoriatic lesions, quantitative PCR of keratinocyte cell line (HaCaT) treated with TGFβ. Moreover, to elucidate how CyA effects on the UPR in keratinocytes, we set out quantitative PCR and western blotting, HaCaT and squamous cell carcinoma cell lines (HSC-1) treated with CyA and CyA analog, cyclosporin D. Furthermore, the siRNA-mediated knockdown effect of cyclophilin (Cyp) A, Cyp B and Cyp C on HaCaT cells were also examined. As a result, the UPR was downregulated in keratinocytes from psoriatic lesions, characterized by immunocytochemical staining of GRP78/Bip, CHOP/GADD153, HRD1 and C/EBPβ. TGFβ induced UPR markers in HaCaT cells. CyA treatment and siRNA-mediated knockdown of Cyp B induced the UPR in HaCaT cells or HSC-1 cells. Altogether, we demonstrate that in psoriasis vulgaris CyA or reduction in Cyp B by RNA interference might induce the UPR in keratinocytes.     

Reciprocal altered expression of T-cadherin and P-cadherin in psoriasis vulgaris

BACKGROUND: The most characteristic change in psoriasis vulgaris is markedly increased, persistent keratinocyte proliferation. The underlying mechanism of excessive epidermal growth is controversial. We previously found and reported that T-cadherin was expressed in keratinocytes and confined to the basal layer of mouse and human skin. Invasive cutaneous squamous cell carcinoma showed a loss of T-cadherin expression. Another study showed that T-cadherin was a negative growth regulator of epidermal growth factor in T-cadherin transfectant neuroblastoma cells. OBJECTIVES: To obtain insight into the role of T-cadherin in keratinocyte proliferation and to investigate further the pathogenesis of psoriasis vulgaris, we examined the expression of T-cadherin, as well as E- and P-cadherin, in psoriasis vulgaris. METHODS: Four untreated active psoriatic skin samples from psoriasis vulgaris patients and four normal human skin samples from plastic surgery were collected, cryosectioned and immunohistochemically stained by antihuman T-, P- and E-cadherin antibodies. Further, the immunofluorescence intensities of T- and P-cadherin on the basal layer of the epidermis were quantitatively measured by the histogram function of LSM 510 software installed in a Zeiss laser scanning confocal microscope. The data were statistically analysed by Student's t-test. RESULTS: It was observed that T-cadherin was weakly and discontinuously expressed on the basal layer of psoriatic skin, while it was intensively expressed on all basal keratinocytes in normal human skin. In contrast, P-cadherin was strongly expressed throughout the entire epidermal layer in psoriatic skin samples, although its expression is restricted to the basal cell layer in normal human skin. There were no obvious differences in E-cadherin expression between normal human skin and psoriatic skin. Statistical analyses showed that the immunofluorescence intensity of T-cadherin in the basal cell layer of psoriatic skin (35 +/- 9.08) was significantly decreased compared with that in normal human skin (131.75 +/- 3.49, P = 2.46 x 10(-6)). There was a significant increase (P = 0.00139) in the immunofluorescence intensity of P-cadherin in the basal layer of psoriatic skin (68.25 +/- 12.13) compared with normal human skin (26 +/- 4.90). CONCLUSIONS: The present study demonstrates that there is downregulation of T-cadherin expression and upregulation of P-cadherin expression in psoriatic skin, which are considered to be involved in the hyperproliferation of keratinocytes in psoriasis vulgaris.     

寻常型银屑病患者皮损中白介素-22和S100A7,A8,A9mRNA的表达及关系

目的研究白介素-22(IL-22)和S100A7,A8,A9mRNA在寻常型银屑病皮损中的表达及关系。方法采用逆转录聚合酶链反应(RT-PCR)技术对35例寻常型银屑病患者皮损和16例正常人皮肤中IL-22,S100A7,A8,A9mR-NA进行检测。结果①IL-22和S100A8,A9mRNA在寻常型银屑病皮损中呈阳性表达,而在正常皮肤中为阴性;S100A7mRNA在寻常型银屑病皮损中的表达水平为1.133±0.0403,较正常对照组0.744±0.0371明显升高,差异有显著性(P<0.01)。②IL-22mRNA与S100A7,A8,A9mRNA的表达呈显著正相关r1=0.543,r2=0.774,r3=0.621,P均<0.01。结论IL-22和S100A7,A8,A9与银屑病的发生和发展中明确相关,可能成为银屑病治疗新的靶位点。     

寻常型银屑病皮损肥大细胞密度及IL-8表达的研究

目的：探讨肥大细胞和IL-8在寻常型银屑病中的作用。方法：采用免疫组化技术(SABC法)观察寻常型银屑病皮损处MC分布和IL-8在表皮中的表达情况。结果：寻常型银屑病皮损处MC密度明显高于皮肤血管炎组和健康对照组；寻常型银屑病皮损处KC中IL-8的表达明显高于两对照组。结论：结果提示MC和IL-8参与寻常型银屑病的致病过程，并且两者之间存在相关性。     

寻常型银屑病皮损中细胞周期蛋白A、D1、B1和E的表达

目的：探讨细胞周期蛋白A（Cyclin A）、Cyclin B1、Cyclin D1和Cyclin E在寻常型银屑病皮损中的表达和意义。方法：采用免疫组化法对28例寻常型银屑病患者的皮损和18例正常人对照皮肤中的Cyclin A、D1、B1和E的表达进行了检测。结果：28例寻常型银屑病患者中26例皮损表达Cyclin A和Cyclin B1，27例有Cyclin D1和Cyclin E表达，正常组织无表达或弱表达。结论：细胞周期蛋白A、D1、B1和E可能通过诱导细胞增殖参与了银屑病的发病。     

c－myc、p53和bcl－2与银屑病角质形成细胞凋亡异常关系的研究

目的 探讨原癌基因ｃ－ｍｙｃ、抑癌基因ｐ５３和凋亡保护基因ｂｃｌ－２在银屑病角质形成细胞凋亡异常中的作用。方法 采用免疫组化和非同位素核酸原位杂交技术从蛋白和ｍＲＮＡ二个水平检测了３６份建党型银屑病皮损中ｃ－ｍｙｃ、ｐ５３和ｂｃｌ－２的表达状况。结果 银屑病表皮中的ｃ－ｍｙｃ、ｐ５３和增殖细胞核抗原（ＰＣＮＡ）阳性细胞较正常皮肤明显增加,而ｂｃｌ－２阳性细胞在表皮基底层中明显减少。在ＰＣＮＡ阳性细