银屑病皮损中IL-20及其受体的表达

目的: 检测IL-20及其受体mRNA在寻常型银屑病(PV)皮损组织中的表达.方法: 采用原位杂交方法检测35例PV皮损和20名正常人皮肤组织IL-20及其受体mRNA的表达.结果: IL-20及其受体mRNA的表达主要表现在PV皮损组织的基底细胞层,阳性率为100%,在正常组织中的表达为阴性或弱阳性,阳性率为10%.结论: IL-20及其受体在PV的发病机制中起重要作用.     

银屑病患者皮损表皮细胞促凋亡分子PDCD5表达的研究

目的 探讨银屑病患者表皮细胞过度增殖与细胞凋亡分化调节异常的关系.方法 寻常性银屑病患者30例,正常人对照28例.采用直接免疫荧光观察促凋亡分子PDCD5的表达状态,流式细胞仪和计算机CELL Quest软件定量分析表皮单细胞表达PDCD5的平均荧光强度和阳性率,逆转录聚合酶链反应方法检测表皮细胞表达PDCD5 mRNA的变化.结果 银屑病患者皮损增生的表皮组织细胞内PDCD5蛋白的表达明显降低,表皮单细胞表达PDCD5促凋亡分子的平均荧光强度和阳性率较正常人显著降低(P<0.01),皮损组织细胞表达PDCD5 mRNA显著低于正常人.结论 银屑病患者局部皮损表皮细胞的过度增殖与促凋亡分子PDCD5表达的下调有关,与表皮细胞的凋亡及终末分化过程的调节异常密切相关.     

角蛋白K6、K16在寻常性银屑病皮损中的表达及其临床意义

目的：探讨角蛋白K6、K16在银屑病发病机制中的地位。方法：用免疫组化法，检测30例银屑病患者皮损处和10例正常对照者皮肤角质形成细胞中角蛋白K6和K16的表达水平。结果：①银屑病组患者皮损处表皮角质形成细胞中K6、K16的表达水平与正常对照组相比，差异具有显著性，其中，病例组棘细胞层K6、K16的表达与对照组相比，差异具有极显著性；②角蛋白K6、K16在银屑病进行期和稳定期的表达差异无显著性，但分别与消退期银屑病相比差异均具有显著性；③角蛋白K6、K16的表达在急性点滴状和慢性斑块状银屑病间差异无显著性。结论：角蛋白K6、K16在银屑病发病机制中可能起重要作用，这两个指标有望作为监测银屑病活动性的指标。     

结合珠蛋白在银屑病皮损及皮损周边外观正常皮肤中的表达

目的 从mRNA及蛋白质水平研究结合珠蛋白在银屑病皮损及皮损周边外观正常皮肤中的表达,探讨其与朗格汉斯细胞的关系及在银屑病发病中的作用.方法采用免疫组化、免疫荧光双标记和原位杂交技术检测银屑病皮损及皮损周边外观正常皮肤中结合珠蛋白的表达.结果与正常人皮肤相比,银屑病皮损处角质形成细胞中结合珠蛋白mRNA的表达均明显增强(P＜0.001);皮损周边外观正常皮肤中的表达与正常人皮肤差异无显著性(P＞0.05).免疫组化显示:皮损处部分角质形成细胞胞浆中有结合珠蛋白表达;皮损及皮损周边外观正常皮肤中均可见结合珠蛋白在部分朗格汉斯细胞中呈阳性表达,且两者中结合珠蛋白阳性朗格汉斯细胞与朗格汉斯细胞总计数的比值较正常皮肤显著增高(P＜0.001).结论银屑病皮损处角质形成细胞中结合珠蛋白mRNA的表达增强,并能合成结合珠蛋白.合成结合珠蛋白的角质形成细胞可能在银屑病发病机理中起负反馈调节作用。     

寻常性银屑病患者皮损及外周血单一核细胞中IL-17A的表达

目的研究Th17细胞及Th17细胞特异性因子IL-17A在寻常性银屑病发病中的作用。方法收集30例寻常性银屑病患者以及18例正常人对照的外周血单一核细胞(PBMC)和皮肤组织,通过实时定量逆转录聚合酶链反应法(RT-PCR)检测IL-17AmRNA的表达水平;免疫组化法分析IL-17A的蛋白表达及IL-17A阳性细胞数百分比。结果银屑病组PBMC与皮损中IL-17AmRNA的相对表达量分别为2.962±1.398和2.023±1.890,对照组分别为0.807±0.839和0.262±0.104,t血IL-17A=-2.133,P<0.05;t皮IL-17A=-2.575,P<0.05;患者组和正常对照PBMC中IL-17A的蛋白表达强度分别为0.467±0.050,0.368±0.065(t=4.495,P<0.05);PBMC中IL-17A+细胞的百分率分别为0.143±0.090和0.090±0.039(t=2.159,P<0.05);患者组皮肤组织IL-17A阳性表达的平均光密度值为0.320±0.038,对照组为0.257±0.041(t=-4.357,P<0.05)。结论银屑病患者外周血及皮损内Thl7细胞因子IL-17A的mRNA与蛋白水平均明显升高,Thl7细胞数目增多,提示其可能与银屑病的发生有一定的相关性。     

β趋化因子CCL20及其受体CCR6mRNA在寻常性银屑病患者皮损中的表达

目的探讨β趋化性细胞因子家族中CC趋化因子配体20(CCL20)及其受体CC趋化因子受体6(CCR6)mRNA在寻常性银屑病患者皮损中的表达情况.方法应用逆转录-聚合酶链式反应(RT-PCR)方法检测35例寻常性银屑病患者皮损和18例正常人皮肤中CCL20和CCR6mRNA的表达.结果与正常人皮肤相比,寻常性银屑病患者皮损中CCL20与CCR6mRNA的表达水平均明显增高(P＜0.001).结论寻常性银屑病患者皮损中CCL20和CCR6 mRNA表达水平的上调可能与银屑病的发病有关.     

MiRNA-203在寻常性银屑病皮损的表达及其对HaCaT细胞增殖的影响

目的 研究微小核糖核酸(miRNA)-203在寻常性银屑病患者皮损中的表达,并探讨对角质形成细胞株(HaCaT细胞)增殖的影响.方法 取2014-2016年23例寻常性银屑病患者的皮损组织和相邻非皮损组织.荧光定量PCR法检测组织中miRNA-203的表达水平,并以5'端、3'端地高辛标记的探针对皮肤组织切片中目的miRNA进行原位杂交,观察miRNA-203在皮肤组织中的定位情况.将miRNA-203模拟物(miRNA-203模拟物组)和miRNA-203模拟物阴性对照(阴性对照组)分别转染HaCaT细胞,正常细胞培养组作为空白对照组,采用噻唑蓝(MTT)法、流式细胞仪和Western印迹法分别对HaCaT细胞的增殖、细胞周期及相关周期蛋白(Cyclin D1、Cyclin B1)的变化进行检测.结果 miRNA-203特异性地表达在表皮的角质形成细胞中,除细胞核外,细胞质亦有表达,且寻常性银屑病患者皮损组织中miRNA-203表达水平(1.35±0.28)显著高于非皮损组织(0.52±0.09),差异有统计学意义(t=6.76,P=0.012).转染miRNA-203模拟物能抑制HaCaT细胞增殖(F=9.36,P=0.007),且空白对照组、阴性对照组和miRNA-203模拟物组HaCaT细胞增殖率均随时间的延长逐渐增加(F=18.68,P＜0.001).与阴性对照组和空白对照组相比,miRNA-203模拟物组HaCaT细胞被阻滞在G2/M期(G2/M期细胞比例:31.33％±4.56％比17.02％±3.53％、16.67％±3.32％,均P＜0.05),HaCaT细胞周期蛋白周期蛋白D1表达水平较高(1.15±0.13比0.52±0.05、0.56±0.07,均P＜0.05),而周期蛋白B1水平较低(0.43±0.08比0.93±0.16、0.91±0.0.15,均P＜0.05).结论 miRNA-203可能参与了寻常性银屑病的发生发展过程.     

脓疱性银屑病患者外周血IL-8水平及其在皮损中的表达

许多学者发现,IL-8与银屑病发病有密切关系,脓疱性银屑病皮损中有比寻常性银屑病皮损中更多的中性粒细胞浸润和IL-8持续升高.2004年7月至2006年2月,我们采用双抗体夹心法及免疫组化技术检测脓疱性银屑病、寻常性银屑病及正常人对照组外周血IL-8水平及皮损处IL-8的表达情况.     

bFGF及其mRNA在银屑病皮损的表达

目的探讨碱性成纤维细胞生长因子(bFGF)在银屑病发病机制中的作用。方法采用组织切片免疫组化和原位杂交技术对22例银屑病皮损和19例正常皮肤的bFGF进行检测。结果寻常型进行期银屑病皮损表皮bF-GF蛋白和mRNA阳性主要表达于表皮基底层和棘层,定位于细胞核、细胞浆和细胞膜;正常皮肤则表达于表皮的基底层,定位于细胞核、细胞浆和细胞膜。统计学分析发现银屑病皮损区表皮与正常皮肤表皮bFGF蛋白和mRNA均有显著性差异(P<0.01)。结论bFGF可能参与银屑病角质形成细胞增殖和真皮微血管异常的发生。     

豚鼠银屑病样动物模型皮损中IL-20的表达水平及反义寡核苷酸基因治疗

目的　研究IL 2 0在豚鼠银屑病样动物模型皮损中的作用。方法　用 5 %心得安乳剂外涂豚鼠耳背部皮肤诱导银屑病样动物模型 ,应用原位杂交及反转录PCR检测皮损中IL 2 0的表达水平。进而观察IL 2 0反义寡核苷酸对皮损的影响。结果　在正常皮肤IL 2 0的表达仅限于基底层 ,且为低表达 ;而实验组皮损中 ,基底层及棘细胞层均有高表达。经过图象扫描后的处理结果显示 ,实验组皮肤中IL 2 0的表达水平较正常对照组显著增高 (t =4.874,P <0 .0 1)。应用IL 2 0反义寡核苷酸乳剂外涂皮损后 ,可使其明显好转。结论　从分子水平进一步证明了豚鼠银屑病样动物模型的可行性 ,同时说明IL 2 0在银屑病样动物模型皮损的形成过程中发挥重要作用。     

银屑病患者皮损Toll样受体4、核因子-κBp65、CC趋化因子配体20的表达

目的 探讨Toll样受体4(TLR4)、核因子(NF)-κBp65、CC趋化因子配体20(CCL20)在银屑病患者皮损区及非皮损区的表达及意义.方法 用免疫组化EnVision法检测40例进行期银屑病患者斑块状皮损及20例进行期银屑病患者非皮损区皮肤、10例正常皮肤石蜡标本中的TLR4、NF-κBp65、CCL20的表达,对其在皮损中的表达水平进行相关性分析.结果 银屑病皮损区角质形成细胞中TLR4、NF-κBp65、CCL20的表达(A值分别为0.3006±0.1200、0.4551±0.1572和0.3862±0.1304)较正常人皮肤TLR4、NF-κBp65、CCL20的表达(A值分别为0.1379±0.0663、0.1970±0.0679和0.1262±0.0612)及银屑病非皮损区组(A值分别为0.1930±0.0687、0.2626±0.0606和0.2387±0.0467)明显上调(t值分别为4.113、5.044、6.106、3.711、5.265和4.889,均P＜0.05).与正常人皮肤相比,银屑病非皮损区TLR4、NF-κBp65、CCL20表达明显上调(t值分别为2.118、2.685和5.608,均P＜0.05).银屑病患者TLR4、NF-κBp65、CCL20的表达水平间均呈正相关(r值分别为0.766、0.851和0.885,均P＜0.05).结论 TLR4、NF-κBp65、CCL20的高表达可能与银屑病发病有关.     

MiR-486-3p在银屑病皮损的表达及对角蛋白17表达的影响

目的 探讨银屑病患者皮损与健康人皮肤中miR-486-3p表达情况及其对角蛋白17（K17）表达的调控作用。 方法　采用生物信息学方法预测出调节K17表达的微RNA（microRNA）,选取10例银屑病患者皮损和10例健康人皮肤提取RNA,用加PolyA尾法逆转录为cDNA,实时荧光定量PCR（qRT-PCR）进行荧光定量。用miR-486-3p模拟物（mimics）和阴性对照物（NC）转染人永生化角质形成细胞（HaCaT细胞）,Western印迹检测K17表达情况;构建双荧光素酶报告系统,其中包括含有K173′UTR的片段,突变1组为将种子序列删除的片段,突变2组为将种子序列间隔突变的片段,突变3组为将种子序列重复2次的片段,并检测miR-486-3p是否与K17 3′UTR种子序列结合而抑制K17表达。 结果　银屑病皮损中miR-486-3P表达水平为0.211 ± 0.120,低于健康对照的0.555 ± 0.425,银屑病皮损为健康对照的0.380,两组比较,t = 2.62,υ = 9,P < 0.05。Western印迹结果显示,miR-486-3p类似物转染HaCaT细胞后,K17表达水平明显降低。双荧光素酶报告系统检测结果,含有K173′UTR种子序列组与突变3组荧光强度分别为65.31 ± 6.32和54.18 ± 10.01,均低于对照组（P < 0.05）;突变1组和突变2组荧光强度分别为114.77 ± 16.14和110.21 ± 12.99,与对照组差异无统计学意义（P > 0.05）。 结论 miR-486-3p 在银屑病皮损中的表达水平低于健康人皮肤,miR-486-3p通过与K17 3′UTR种子序列结合抑制K17表达,提示其表达水平降低及对K17调控作用的改变可能与银屑病的发生与发展相关。     

miR-320及下游靶基因survivin在银屑病皮损中的表达

目的检测寻常性银屑病皮损中miR-320及其下游靶基因survivin mRNA的表达水平。方法采用实时定量聚合酶链反应(RT-PCR)检测40例寻常性银屑病患者皮损及40名健康对照皮肤中miR-320、survivin mRNA的表达水平,运用双荧光素酶报告实验验证survivin是miR-320的靶基因。miR-320和survivin表达水平两组间比较采用t检验,寻常性银屑病皮损中miR-320与survivin表达的相关性采用Pearson相关分析。结果与健康对照皮肤相比,寻常性银屑病皮损中miR-320表达量为对照组的(0.561±0.11)倍,表达明显下调(t=3.06,P0.05);survivin mRNA表达量为对照组的(2.034±0.26)倍,表达水平明显上调(t=3.35,P0.05);双荧光素酶报告实验结果提示survivin是miR-320的靶基因,寻常性银屑病皮损中miR-320与survivin mRNA呈负相关(r2=0.634,P0.05)。结论 miR-320可能通过调控其下游靶基因survivin的异常表达参与寻常性银屑病皮损的形成过程。     

Immunohistochemical analysis of sensory nerves and neuropeptides,and their contacts with mast cells in developing and mature psoriatic lesions

The distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) was studied immunohistochemically in psoriatic skin during the Koebner response (6 h, 2 days, 7 days, 14 days, 21 days), and in mature psoriatic plaques, of 37 psoriatic patients. The morphological association of sensory nerves, SP and VIP with papillary mast cells was also monitored. The nerves containing SP, VIP or CGRP were very scanty in control skin, and in non-lesional and Koebner-negative psoriatic skin. The first psoriatic lesions were seen 7 days after tape stripping the symptomless psoriatic skin. SP- and VIP-containing nerves were slightly increased in Koebner-positive specimens, but the increase was very prominent in dermal papillae of mature psoriatic plaques. In the plaques, nerve-mast cell contacts were significantly increased (p<0.001) compared with non-lesional psoriatic skin. Only SP-positive fibres were detected in the epidermis and in contact with papillary mast cells. VIP was mainly located around capillaries where SP was also found. No change was noted in CGRP-positive fibres between lesional and non-lesional specimens. The appearance of SP and VIP in the capillary walls is morphological evidence for their function as vasodilators in psoriatic lesion. A slight increase in SP- and VIP-positive fibres in Koebner-positive specimens suggests that these neuropeptides may participate in the inflammatory reaction at an early stage. Their prominence in mature psoriatic plaques in turn indicates a role for them in the maintenance of psoriatic lesions. Morphological contacts between mast cells and SP-containing nerves give further evidence to the view that SP is capable of amplifying the inflammatory reaction also through the axon-reflex mechanism.Part of this work was presented at the meeting of the European Society for Dermatological Research, London, UK, 4–7 April 1992     

Mast cell tryptase and chymase in developing and mature psoriatic lesions

The number and distribution of mast cells in nonlesional and lesional skin samples from 13 psoriatic patients were analyzed enzyme- and immunohistochemically. Mast cell tryptase was stained with the sensitive substrate Z-Gly-Pro-Arg-4-methoxy-2-naphthylamide, and chymase with Suc-Val-Pro-Phe-MNA and monoclonal B7 anti-chymase antibody. In addition, healthy-looking skin from 27 psoriatic patients was tape-stripped resulting in induction of the Köbner response in 9 patients. Sequential biopsies were taken before and after (7, 14 and 21 days) tape-stripping, and both tryptase and chymase were stained enzyme-histochemically. In nonlesional psoriatic skin, 70 ± 24% (mean ± SD) of the mast cells contained chymase enzyme activity, and 78 ± 18% chymase immunoreactivity. About 10% of the chymase-immunoreactive cells lacked chymase activity. In lesional psoriatic skin, tryptase-positive cells were increased in number throughout the dermis but especially beneath the epidermis. Chymase immunoreactivity paralleled the tryptase activity, whereas chymase activity was strongly diminished both in terms of mast cell numbers and in staining intensity in the papillary dermis. The apparent inactivation of chymase may be due to the action of the chymase inhibitors, ₁-antitrypsin and ₁-antichymotrypsin, localized immunohistochemically in mast cells of lesional and nonlesional psoriatic skin. In the developing psoriatic lesion, mast cells displaying chymase activity were already 27–38% decreased in number in the upper dermis on day 7 after tape-stripping, along with the first clinical signs of psoriasis. Earliest alterations in tryptase-positive cells were observed on day 14 as increased mast cell contacts with the epidermis combined with only a slight increase in mast cell numbers in the upper dermis. During the development of a psoriatic lesion, TC mast cells (tryptase⁺, chymase⁺) increase in number in the upper dermis, but chymase becomes inactive at an early stage. The abundant presence of active trypase but inactive chymase in the upper dermis may have a potential role in psoriasis since both of these enzymes can process several biologically active peptides and proteins.     

CCL20、CCR6及IL-17A在寻常型银屑病患者血清中的表达及意义

目的观察趋化因子CCL20及其受体CCR6、白细胞介素(IL)-17A在银屑病患者血清中的变化,并探讨其在银屑病发病中的作用。方法采用酶联免疫吸附法测定26例斑块型、7例点滴型银屑病患者及8名健康人血清中CCL20、CCR6及IL-17A的浓度,并对银屑病皮损面积和严重程度指数(PASI)评分、病程进行相关性分析。结果斑块型银屑病患者血清CCL20、CCR6及IL-17A浓度(748.74±268.72,10.20±3.75,39.22±13.21)均显著高于健康对照组(578.45±204.93,6.79±4.61,25.54±13.04)(P分别0.05,0.01,0.01)。点滴型银屑病与对照组差异无统计学意义。CCL20、CCR6与PASI评分呈正相关(r=0.416,r=0.350)(P0.05)。CCL20、CCR6及IL-17A三者之间均存在显著正相关(r=0.852,r=0.801,r=0.825)(P0.001),与病程无明显相关性。结论 CCL20、CCR6及IL-17A参与斑块型银屑病的病理过程。     

窄谱中波紫外线治疗前后银屑病皮损中CD1a^＋DC、Ki-67的表达

目的探讨窄谱中波紫外线（NB—UVB）治疗对银屑病患者皮损中CD1a^＋树突细胞（CD1a^＋DC）及Ki-67水平的影响。方法采用免疫组化法分析寻常性银屑病患者NB—UVB治疗前后皮损中及非皮损中CD1a^＋DC及Ki-67的表达。采用银屑病皮损面积和严重性指数（PASI）进行病情严重程度评分。结果27例银屑病患者中,治疗前皮损中CD1a^＋DC及Ki-67表达水平（15±4;53±12）高于非皮损（9±3;22±6）及治疗后患者皮损的表达量（9±3;25±1）（P〈0．05）。银屑病皮损的严重程度PASI评分与Ki-67表达相关（r=0．29,P〈0．05）。银屑病皮损的严重程度PASI评分与CD1a抗原的表达量之间无相关性（r=-0．27,P〉0．05）。结论NB—UVB可以通过调节皮肤免疫系统达到治疗目的。银屑病患者皮损中Ki-67表达水平与银屑病皮损的严重程度PASI评分有关。     

The presence of tryptase-positive and bikunin-negative mast cells in psoriatic skin lesions

Human mast cells are well known to produce a serine protease, tryptase, which appears to play a pathogenic role in various skin inflammations. It was previously reported that a rat homologue of bikunin may inhibit tryptase activity. Various type of cells (i.e. keratinocytes) are able to produce this protein inhibitor, it still remains unclear if bikunin is present in dermal inflammatory milieu, in which mast cells, through secretion of tryptase, play an inflammatory role. Therefore, the purpose of the present study was to exploit expression and production of bikunin in dermis and dermal constituents. We first compared the dermal mast cells in psoriatic lesions with those in lesional skin of atopic dermatitis or of chronic eczema by use of immunoelectron microscopy and immunohistochemical analyses using antibodies to bikunin and tryptase. Then, we tested what kinds of cytokines may regulate the de novo synthesis of bikunin. To do so, RNA was extracted from a human mastocytic cell line, HMC-1, reverse-transcribed, and semiquantitative RT-PCR was performed using primers specific for bikunin. With immunoelectron microscopy, bikunin was found to localize on the cell membrane, while tryptase was in the secretary granules of the mast cells. In psoriatic lesions, around 70% of dermal mast cells were positive for both tryptase and bikunin, and the remaining was mostly positive for tryptase, but the expression of bikunin was under the detection limit of the experimental setting. This observation was seen in only psoriatic lesions, even in almost cured lesions, while in atopic dermatitis or chronic eczema only mast cells doubly positive for bikuin and tryptase were seen. In HMC-1, bikunin was constitutively expressed at an mRNA level, which was upregulated by stimulation with interleukine-4, but was suppressed by interferon-γ. Bearing in mind the concept that in psoriasis local cytokine milieu is shifted toward a Th1 pattern (predominant secretion of interferon-γ), tryptase-positive, bikunin-negative mast cells may be induced.     

银屑病患者皮损间充质干细胞环状RNA表达异常研究

目的 探讨环状RNA（circRNA）在银屑病发病中的作用。方法 分离、培养15例银屑病患者皮损及15例健康对照皮肤间充质干细胞（MSC）,用流式细胞仪及多向分化法进行鉴定。用RNA测序法检测circRNA表达,并进行详细的生物信息学分析。挑选7个差异表达的circRNA构建circRNA-microRNA相互作用网络,从中挑选3个与其相关的microRNA进行qRT-PCR验证。银屑病患者组和对照组qRT-PCR验证结果采用两独立样本t检验。结果 RNA测序结果显示,共检测到6 323个circRNA,其中3 227个为本实验首次发现。与对照组相比,患者组中有129个circRNA呈差异表达,其中123个表达上调,6个表达下调。挑选7个差异circRNA进行circRNA-microRNA相互作用预测,显示与这7个circRNA关系密切的银屑病相关microRNA,包括miR-17-5p、miR-30e-5p、miR-142-3p/5p、miR-369-3p、miR-184、miR-4490、miR-654-3p、miR-423-5p等。qRT-PCR也证实,这7个差异circRNA在患者组也表达上调。与对照组相比,挑选的3个microRNA在银屑病皮损中低表达（t值分别为3.993、3.217、2.918,均P < 0.05）。结论 银屑病皮损MSC circRNA表达异常,并可能参与了银屑病发病。