需要JoVE订阅才能观看此内容。 请登录或开始免费试用

方法文章

利用细胞因子联合刺激HaCaT细胞建立体外银屑病样皮肤转录谱

5K 次观看

⸱

DOI：

10.3791/61537

⸱

2021年3月15日

 ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University and Collaborative Innovation Center for Biotherapy, 2Department of Cardiovascular Medicine, West China Hospital, Sichuan University

* These authors contributed equally

本文内容

摘要

本文介绍了一种在HaCaT细胞系上联合使用五种细胞因子（IL-17A、IL-22、IL-1α、TNF-α、OSM）在转录水平建立体外银屑病样皮肤炎症模型的方法。

摘要

银屑病是一种由先天性和适应性免疫系统介导的常见慢性炎症性皮肤病，其特征是表皮角质形成细胞的异常增殖与分化，以及炎性细胞的浸润。皮肤特异性的角质形成细胞是先天免疫的关键参与者，能够响应免疫细胞和外界刺激，从而在银屑病的免疫病理发生过程中发挥重要作用。本文介绍一种利用五种促炎细胞因子组合（M5组合）在转录水平上诱导HaCaT细胞系产生银屑病样角质形成细胞炎症的方法，该组合包括IL-17A、IL-22、IL-1α、TNF-α和抑癌蛋白M。结果显示，经M5组合处理的HaCaT细胞中抗菌肽（BD2、S100A7、S100A8和S100A9）、趋化因子及细胞因子（CXCL1、CXCL2、CXCL8、CCL20、IL-1β、IL-6和IL-18）的表达水平均升高。角质形成细胞分化标志物（Keratin1、Keratin10、Filaggrin和Loricrin）的mRNA水平则下调，这与来自银屑病样角质形成细胞的转录组数据一致。因此，本方法在体外建立了转录水平上的银屑病样皮肤炎症模型，有助于银屑病分子发病机制的研究。

引言

银屑病是一种常见的非传染性慢性炎症性皮肤病，由免疫反应失调引发，主要影响构成表皮的角质形成细胞1，其特征是角质形成细胞异常快速增殖，并伴有角化过度和角化不全。全球约有3%的人口受此疾病影响2。该病还常伴随多种共病，进一步加重疾病负担，包括由该综合征引起的心血管疾病和代谢综合征3。

表皮由五层角质形成细胞构成，并随着分化过程发生形态学变化：从内到外依次为基底层、棘层、颗粒层、透明层（存在于手掌和脚底）以及角质层4。表皮分化的改变会导致皮肤屏障功能受损，这在皮肤炎症性疾病的发病机制中具有重要意义5,6,7,8。角质形成细胞在维持完整表皮屏障方面发挥关键作用，可防止水分流失以及抵御紫外线B照射、过敏原和病原体等环境刺激因素9。健康个体中，基底细胞增殖与角质层脱屑之间保持平衡；而包括银屑病在内的多种皮肤疾病则表现为这一复杂机制的失衡10。

除了形成屏障功能外，角质形成细胞也是皮肤免疫系统的关键组成部分。在银屑病的免疫致病机制中，皮肤常驻的1型辅助性T细胞（Th1）和17型辅助性T细胞（Th17）被激活，分别导致IFN-γ和IL-17A的产生增加。这些细胞因子可诱导角质形成细胞中趋化因子（CCL20，CXCL1/2/8/9/10/11）、抗菌肽（BD2，LL37，S100A7/8/9/12）及其他炎症因子（TNF-α，IL-6，IFN-β）的合成增加，从而招募更多的Th1细胞、Th17细胞和中性粒细胞进入皮肤，进一步放大IL-17/IL-23轴的作用11。角质形成细胞与免疫细胞之间的相互作用是银屑病发生和维持的原因11。

已有研究描述了银屑病中复杂的细胞因子网络，并强调了免疫细胞浸润产生的促炎性细胞因子（如 IL-23、IL-22、IL-17、IL-1α、抑癌蛋白 M（OSM）和 TNF-α）所起的核心作用12,13。事实上，既往研究表明，IL-17A、IL-22、IL-1α、TNF-α 和 OSM 水平的升高可在体外诱导正常人表皮角质形成细胞呈现银屑病样表型14。

与原代角质形成细胞相比，永生化角质形成细胞系（HaCaT）更易于获取和培养，且具有更好的可重复性，因此已被广泛用于银屑病的研究15,16,17,18,19,20。与人乳头瘤病毒16型E6/E7转化的HEK001和KerTr细胞不同，HaCaT细胞系能够表达分化相关的基因产物，包括Keratin1（KRT1）、Keratin10（KRT10）、Loricrin和filaggrin20,21,22，因而成为研究角蛋白化及促炎反应调控机制的一种接近原代角质形成细胞的理想工具。

KRT5/14 是增殖性基底角质形成细胞中主要表达的I型-II型角蛋白对，而表皮上层的分化角质形成细胞则下调 KRT5/14 的表达，并转而表达 KRT1/10 作为主要的角蛋白对23。与健康皮肤相比，银屑病皮损中角蛋白表达的变化包括 KRT1/10 表达降低24,25，以及银屑病表皮中 KRT5/14 表达升高26，这些变化特征表现为表皮过度增殖和角化不全27。兜甲蛋白（Loricrin）是一种终末分化结构蛋白，占角化包膜的70%以上，有助于维持角质层的保护屏障功能28，但在银屑病患者皮肤中其表达下调29。丝聚蛋白（Filaggrin）在角质形成细胞分化的最后阶段表达，参与类似支架结构的角化包膜的聚集30，在银屑病皮损皮肤中其表达减少29。

总体而言，我们的目标是利用细胞因子组合在HaCaT细胞中建立炎症性角质形成细胞模型，该模型能够协同模拟银屑病皮损的某些特征，包括启动免疫反应、角质形成细胞增殖与分化。

访问受限。请登录或开始试用以查看此内容。

方案

在无菌条件下进行步骤1至3。所有培养基均含有0.1 mg/mL的青霉素和链霉素。

1. 细胞制备

  1. 在100 mm细胞培养皿中加入10 mL含10%胎牛血清（FBS）的杜尔贝科改良伊格尔培养基（DMEM），接种1 × 106个HaCaT细胞。将培养皿置于37 °C、含5% CO2的湿润培养箱中培养2天。
  2. 当细胞培养至约80%融合度时，小心吸除培养皿中的培养基，并用5 mL 1×磷酸盐缓冲液（PBS）洗涤细胞。手动轻轻晃动培养皿。
  3. 吸除PBS，向细胞中加入2 mL 0.25%胰蛋白酶-EDTA溶液。轻轻振荡培养皿，使溶液充分覆盖细胞单层。
  4. 将细胞置于37 °C、含5% CO2的湿润培养箱中孵育5分钟，直至相差显微镜下观察到细胞明显从培养皿表面脱落。
    注意：细胞形态应呈圆形。若细胞未充分脱落，可继续孵育5分钟并辅以手动震荡。
  5. 细胞脱落后，加入5 mL含10% FBS的DMEM以中和胰蛋白酶活性，并用移液器将细胞悬液转移至50 mL离心管中。
  6. 将含有细胞悬液的离心管在180 × g 条件下离心5分钟，弃去上清液。
  7. 向细胞沉淀中加入10 mL含10% FBS的DMEM培养基，轻轻吹打混匀，使细胞充分重悬。

2. 在6孔板中接种细胞

  1. 以每孔 2.0 × 105 个细胞的密度将细胞接种于 6 孔培养板中。
  2. 在 M5 联合刺激前，将 6 孔培养板置于 37 °C、含 5% CO2 的湿润培养箱中孵育过夜，使细胞贴壁。

3. M5 对 HaCaT 细胞的刺激作用

  1. 制备含有重组 IL-17A、IL-22、IL-1α、TNF-α 和 oncostatin M 蛋白的 M5 细胞因子组合混合培养基，各因子浓度均为 10 ng/mL，溶于含 2% FBS 的 DMEM 中。
    注意：M5 组合由 IL-17A、IL-22、IL-1α、TNF-α 和 oncostatin M 重组蛋白组成。M5 组合的协同作用可模拟银屑病的部分特征，例如趋化因子和抗菌肽表达的上调31。
  2. 细胞过夜培养后，弃去上清液，每孔加入 2 mL M5 组合混合培养基。
  3. 继续培养细胞；于 24 小时收集细胞裂解物用于 mRNA 定量分析（步骤 4），或于 48–72 小时收集培养上清液，通过 ELISA 检测细胞因子水平32,33,34,35,36,37（步骤 6）。

4. 收获M5刺激的HaCaT细胞的mRNA

  1. 吸弃 M5 完全培养基，并用 1-2 mL 冷 PBS 洗涤细胞一次。
  2. 吸弃 PBS，直接向培养皿中加入 1 mL 商业化的异硫氰酸胍溶液以裂解细胞。
  3. 用移液器将裂解液上下吹打数次以充分匀浆，然后将细胞裂解液转移至 1.5 mL 微量离心管中，室温放置 5 分钟。
  4. 加入 200 µL 氯仿，剧烈振荡试管约 20 秒，随后在室温下孵育样品 5 分钟。
  5. 在 4 °C 下以 12,000 × g 离心 10 分钟。
  6. 将水相转移至新的 1.5 mL 微量离心管中。
  7. 向水相中加入 500 µL 异丙醇，轻轻混匀，室温静置 5 分钟。
  8. 在 4 °C 下以 10,000 × g 离心 15 分钟。
  9. 吸弃异丙醇，加入 1 mL 75% 乙醇，洗涤沉淀一次。
  10. 在 4 °C 下以 7,500 × g 离心 5 分钟，倒出乙醇，让沉淀在空气中自然干燥。
  11. 向 RNA 沉淀中加入 30 µL 经 DEPC 处理的水，用于后续 RT-PCR 检测。

5. 通过实时 PCR 分析 mRNA 表达

  1. 使用市售试剂盒，按照制造商说明书，以1 µg总RNA进行cDNA合成。
    注意：SYBR Green RT-PCR实验使用1 µg总RNA。
  2. 配制PCR反应体系：12.5 µL SYBR Premix Ex Taq II、1 µL PCR上游引物（10 µM）、1 µL PCR下游引物（10 µM）、2 µg cDNA和8.5 µL dH2O。每反应终体积为25 µL。
    注意：本研究中用于RT-PCR分析的基因引物序列列于表1中。
  3. 在热循环仪中孵育。循环条件包括：95 °C变性30秒，随后进行40个循环（95 °C 5秒、60 °C 30秒、72 °C 20秒），最后进行熔解曲线分析。
  4. 通过2-ΔΔCT法计算Ct值（阈值循环）之间的相对差异，分析RT-PCR实验数据。以β-actin作为内参对照，用于RT-PCR相对定量基因表达的标准化。

6. 收集细胞培养上清用于ELISA

  1. 将每种细胞培养基用移液器转移至1.5 mL微量离心管中。
  2. 在4 °C条件下，以1500 × g 离心10分钟。
  3. 将上清液分装后立即于-80 °C保存。

7. 采用ELISA法分析细胞因子表达

  1. 按照制造商说明准备所有试剂、工作标准品和样品。使用样本稀释液对标准品进行3倍系列稀释，浓度范围为2,000至2.74 pg/mL。用样本稀释液将样品按1:2比例稀释。
  2. 每孔加入100 µL标准品或样品。盖上盖板膜并用石蜡膜密封。室温孵育2.5小时。
  3. 孵育2.5小时后，弃去孔内溶液。每孔加入300 µL洗涤缓冲液，静置3分钟，然后吸去液体。重复此步骤共五次。最后一次洗涤后，将酶标板倒置并在吸水纸上轻拍，以去除残留液体。
  4. 每孔加入100 µL检测抗体溶液。在37 °C下置于摇床上孵育2小时。
    注：生物素标记的检测抗体溶液由各ELISA试剂盒提供。
  5. 吸去孔内溶液，使用300 µL洗涤缓冲液每孔洗涤五次。最后一次洗涤后，将酶标板倒置并在吸水纸上轻拍，以去除残留液体。
  6. 每孔加入100 µL HRP-链霉亲和素结合物。室温下轻轻振荡孵育45分钟。
  7. 孵育结束后，用移液器吸弃孔内溶液。使用300 µL洗涤缓冲液每孔洗涤五次。最后一次洗涤后，将酶标板倒置并在吸水纸上轻拍，以去除残留液体。
  8. 每孔加入100 µL TMB底物溶液进行显色。在37 °C避光孵育30分钟。
  9. 当颜色显现后，每孔加入50 µL终止液。轻轻拍打酶标板以确保充分混匀。立即在450 nm波长下读取吸光度。

访问受限。请登录或开始试用以查看此内容。

结果

M5联合刺激诱导HaCaT细胞的炎症反应。
HaCaT细胞经M5细胞因子组合刺激或不刺激处理24小时，检测与银屑病相关的基因mRNA表达水平，这些基因参与免疫和炎症趋化因子及抗菌肽的调控。与未处理的HaCaT细胞相比，经M5联合刺激的HaCaT细胞中，中性粒细胞趋化因子CXCL138、CXCL239、CXCL840以及T细胞趋化因子CCL2041,42的表达显著升高，且呈时间依赖性（图1A）。抗菌肽BD243、S100A7...

访问受限。请登录或开始试用以查看此内容。

讨论

本文描述了一种利用五种细胞因子组合（IL-17A、IL-22、IL-1α、TNF-α、OSM）作用于HaCaT细胞系，以在转录水平上建立体外类银屑病皮肤炎症谱的方法。该方案可适用于研究基因在银屑病发病机制中的作用，以及用于银屑病治疗药物的筛选。近期研究表明，病变皮肤中产生IL-17A和IL-22的CD8 T细胞过度表达，提示其参与了银屑病的发病过程61。IL-1α、IL-22和IL-17A可在动物模型中诱导皮肤炎症62,63,64,65。某些细胞因子能够诱导与抗菌肽产生相关的特异性表达模式，例如IL-17和IL-2266，或与先天免疫反应相关，例如IL-17、IL-1α和TNF-α11,

访问受限。请登录或开始试用以查看此内容。

披露

作者声明无潜在利益冲突。

致谢

本工作受到国家自然科学基金 [81703132、31271483、81472650、81673061、81573050、31872739 和 81601462] 的资助

访问受限。请登录或开始试用以查看此内容。

材料

本文使用的材料清单
姓名公司目录编号评论
DMEM—达尔伯克改良伊格尔培养基Gibco11965092
胎牛血清Gibco10100139C
HaCaT 细胞中国典型培养物保藏中心GDC0106传代次数少于 15 代
人 IL-1β ELISA 试剂盒BeyotimePI305
人 IL-6 ELISA 试剂盒BeyotimePI330
人 IL-8 ELISA 试剂盒BeyotimePI640
IL-1 alpha 人源ProspecCYT-253重组蛋白
IL-17 人源ProspecCYT-250重组蛋白
IL-22 人源ProspecCYT-328重组蛋白
OSM 人源ProspecCYT-231重组蛋白
PBSGibco10010049pH 7.4
青霉素-链霉素Gibco15140163
PrimeScrip 反转录试剂盒TAKARARR047A
TB Green Premix Ex TaqTAKARARR420A
TNF alpha 人源ProspecCYT-223重组蛋白
TRIzo 试剂Invitrogen15596018
胰蛋白酶-EDTA (0.25%)，含酚红Gibco25200072

参考文献

  1. Lowes, M. A., Bowcock, A. M., Krueger, J. G. Pathogenesis and therapy of psoriasis. Nature. 445 (7130), 866-873 (2007).
  2. Gupta, R., Debbaneh, M. G., Liao, W. Genetic epidemiology of psoriasis. Current dermatology reports. 3 (1), 61-78 (2014).
  3. Boehncke, W. -H., Schön, M. P. Psoriasis. Lancet. 386 (9997), London, England. 983-994 (2015).
  4. Zaidi, Z., Lanigan, S. W. Dermatology in Clinical Practice. Lanigan, W. S., Zaidi, Z. , Springer. London. 1-15 (2010).
  5. Proksch, E., Brandner, J. M., Jensen, J. M. The skin: An indispensable barrier. Experimental Dermatology. 17 (12), 1063-1072 (2008).
  6. Van Smeden, J., Janssens, M., Gooris, G., Bouwstra, J. The important role of stratum corneum lipids for the cutaneous barrier function. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids. 1841 (3), 295-313 (2014).
  7. Wolf, R., Orion, E., Ruocco, E., Ruocco, V. Abnormal epidermal barrier in the pathogenesis of psoriasis. Clinics in Dermatology. 30 (3), 323-328 (2012).
  8. Proksch, E., Brasch, J. Abnormal epidermal barrier in the pathogenesis of contact dermatitis. Clinics in Dermatology. 30 (3), 335-344 (2012).
  9. Bäsler, K., Brandner, J. M. Tight junctions in skin inflammation. Pflügers Archiv-European Journal of Physiology. 469 (1), 3-14 (2017).
  10. Hoffjan, S., Stemmler, S. On the role of the epidermal differentiation complex in ichthyosis vulgaris, atopic dermatitis and psoriasis. British Journal of Dermatology. 157 (3), 441-449 (2007).
  11. Lowes, M. A., Russell, C. B., Martin, D. A., Towne, J. E., Krueger, J. G. The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses. Trends in Immunology. 34 (4), 174-181 (2013).
  12. Saxena, A., Raychaudhuri, S. K., Raychaudhuri, S. P. Psoriatic Arthritis and Psoriasis. , Springer. 73-82 (2016).
  13. Lowes, M. A., Suárez-Fariñas, M., Krueger, J. G. Immunology of psoriasis. Annual Review of Immunology. 32, 227-255 (2014).
  14. Guilloteau, K., et al. Skin inflammation induced by the synergistic action of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α recapitulates some features of psoriasis. The Journal of Immunology. 184 (9), 5263-5270 (2010).
  15. Wang, Z., et al. Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation. Autophagy. , 1-24 (2020).
  16. Choi, D. H., Hwang, H. S. Anti-inflammation activity of brazilin in TNF-α induced human psoriasis dermatitis skin model. Applied Biological Chemistry. 62 (1), 46(2019).
  17. Teng, X., et al. IL-37 ameliorates the inflammatory process in psoriasis by suppressing proinflammatory cytokine production. Journal of Immunology. 192 (4), 1815-1823 (2014).
  18. Wu, S., et al. The potential of Diosgenin in treating psoriasis: Studies from HaCaT keratinocytes and imiquimod-induced murine model. Life Sciences. 241, 117115(2020).
  19. Katarzyna, B., Elwira, S., Marta, M., Joanna, J. -B., Magdalena, G. -C. Models in the Research Process of Psoriasis. International Journal of Molecular Ences. 18 (12), 2514(2017).
  20. Auewarakul, P., Gissmann, L., Cid-Arregui, A. Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors. Molecular and Cellular Biology. 14 (12), 8250-8258 (1994).
  21. Boukamp, P., et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. Journal of Cell Biology. 106 (3), 761-771 (1988).
  22. Irma, C., et al. HaCaT cells as a reliable in vitro differentiation model to dissect the inflammatory/repair response of human keratinocytes. Mediators of Inflammation. 2017, 7435621(2017).
  23. Melino, G., et al. The cornified envelope: a model of cell death in the skin. Results & Problems in Cell Differentiation. 24 (4), 175(1998).
  24. Thewes, M., Stadler, R., Mischke, B. K. Normal psoriatic epidermis expression of hyperproliferation-associated keratins. Archives of Dermatological Research. , (1991).
  25. Ishida-Yamamoto, A., Senshu, T., Takahashi, H., Akiyama, K., Iizuka, H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis. Journal of investigative Dermatology. 114 (4), 701-705 (2000).
  26. Elango, T., et al. Methotrexate normalized keratinocyte activation cycle by overturning abnormal keratins as well as deregulated inflammatory mediators in psoriatic patients. Clinica Chimica Acta. , 329-337 (2015).
  27. Ghadially, R., Reed, J. T., Elias, P. M. Stratum corneum structure and function correlates with phenotype in psoriasis. Journal of investigative dermatology. 107 (4), 558-564 (1996).
  28. Nithya, S., Radhika, T., Jeddy, N. Loricrin - An overview. Journal of Oral and Maxillofacial Pathology. 19 (1), (2015).
  29. Kim, B. E., et al. TNF-α downregulates filaggrin and loricrin through c-Jun N-terminal Kinase: Role for TNF-α antagonists to improve skin barrier. Journal of Allergy & Clinical Immunology. 127 (2), (2011).
  30. Gutowska-Owsiak, D., et al. IL-17 downregulates filaggrin and affects keratinocyte expression of genes associated with cellular adhesion. Experimental dermatology. 21 (2), 104-110 (2012).
  31. Guilloteau, K., et al. Skin inflammation induced by the synergistic action of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α recapitulates some features of psoriasis. Journal of Immunology. 184 (9), 5263-5270 (2010).
  32. Jeon, B., et al. Optimization and validation of a method to identify skin sensitization hazards using IL-1 α and IL-6 secretion from HaCaT. Toxicology In Vitro : An International Journal Published in Association with BIBRA. 61, 104589(2019).
  33. Lee, K. -M., Kang, J. H., Yun, M., Lee, S. -B. Quercetin inhibits the poly(dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed human keratinocytes. Biochemical and Biophysical Research Communications. 503 (1), 116-122 (2018).
  34. Lee, N., Chung, Y. C., Kang, C. I., Park, S. -M., Hyun, C. -G. 7,8-dimethoxycoumarin attenuates the expression of IL-6, IL-8, and CCL2/MCP-1 in TNF-α-treated HaCaT cells by potentially targeting the NF-κB and MAPK pathways. Cosmetics. 6 (3), (2019).
  35. Smolińska, E., Moskot, M., Jakóbkiewicz-Banecka, J., Wgrzyn, G., Gabig-Cimińska, M. Molecular action of isoflavone genistein in the human epithelial cell line HaCaT. PLoS One. 13 (2), 0192297(2018).
  36. Ying, X., Yan, Y., Li, Y. Tripterine alleviates LPS-induced inflammatory injury by up-regulation of miR-146a in HaCaT cells. Biomedicine & Pharmacotherapy. 105, 798(2018).
  37. Zhang, M., et al. AIM2 inflammasome mediates Arsenic-induced secretion of IL-1 β and IL-18. Oncoimmunology. 5 (6), 1160182(2016).
  38. Lowes, M. A., et al. Immunology of Psoriasis. Annual Review of Immunology. 32, 227-255 (2014).
  39. Nedoszytko, B., Wojdyło, M. S., Dziurdzińska, K. -S., Roszkiewicz, J., Nowicki, R. J. Chemokines and cytokines network in the pathogenesis of the inflammatory skin diseases: atopic dermatitis, psoriasis and skin mastocytosis. Postepy Dermatologii i Alergologii. 31, 84-91 (2014).
  40. Glowacka, E., Lewkowicz, P., Rotsztejn, H., Zalewska, A. IL-8, IL-12 and IL-10 cytokines generation by neutrophils, fibroblasts and neutrophils- fibroblasts interaction in psoriasis. Advances in Medical Sciences. 55 (2), 254-260 (2010).
  41. Li, H., et al. Cyr61/CCN1 induces CCL20 production by keratinocyte via activating p38 and JNK/AP-1 pathway in psoriasis. Journal of Dermatological Science. 88 (1), 46-56 (2017).
  42. Kim, T. G., et al. Dermal clusters of mature dendritic cells and T cells are associated with the CCL20/CCR6 chemokine system in chronic psoriasis. Journal of Investigative Dermatology. 134 (5), 1462-1465 (2014).
  43. Kolbinger, F., et al. β-Defensin 2 is a responsive biomarker of IL-17A-driven skin pathology in patients with psoriasis. The Journal of Allergy and Clinical Immunology. 139 (3), (2017).
  44. Ekman, A., Vegfors, J., Eding, C., Enerbäck, C. Overexpression of psoriasin (S100A7) contributes to dysregulated differentiation in psoriasis. Acta Dermato-Venereologica. 97 (4), 441-448 (2016).
  45. Peric, M., et al. Vitamin D analog calcipotriol suppresses the Th17 cytokine-induced proinflammatory S100 "Alarmins" psoriasin (S100A7) and Koebnerisin (S100A15) in psoriasis. Journal of Investigative Dermatology. 132 (5), 1416-1424 (2012).
  46. Aochi, S., et al. Markedly elevated serum levels of calcium-binding S100A8/A9 proteins in psoriatic arthritis are due to activated monocytes/macrophages. Journal of the American Academy of Dermatology. 64 (5), 879-887 (2011).
  47. Krueger, J. G., et al. IL-17A is essential for cell activation and inflammatory gene circuits in subjects with psoriasis. Journal of Allergy Clinical Immunology. 130 (1), 145-154 (2012).
  48. Wilsmann-Theis, D., et al. Among the S100 proteins, S100A12 is the most significant marker for psoriasis disease activity. Journal of the European Academy of Dermatology & Venereology. 30 (7), 1165-1170 (2016).
  49. Wang, Z., et al. Systematic screening and identification of novel psoriasis-specific genes from the transcriptome of psoriasis-like keratinocytes. Molecular Medicine Reports. 19 (3), 1529-1542 (2018).
  50. Mizutani, H., Ohmoto, Y., Mizutani, T., Murata, M., Shimizu, M. Role of increased production of monocytes TNF-alpha, IL-1beta and IL-6 in psoriasis: relation to focal infection, disease activity and responses to treatments. Journal of dermatological science. 14 (2), 145-153 (1997).
  51. Pietrzak, A., Lecewicz-Torun, B., Chodorowska, G., Rolinski, J. Interleukin-18 levels in the plasma of psoriatic patients correlate with the extent of skin lesions and the PASI score. Acta Dermato-Venereologica. 83 (4), 262-265 (2003).
  52. Cai, Y., et al. A critical role of the IL-1β-IL-1R signaling pathway in skin inflammation and psoriasis pathogenesis. Journal of investigative Dermatology. 139 (1), 146-156 (2018).
  53. Arican, O., Aral, M., Sasmaz, S., Ciragil, P. Serum levels of TNF-α, IFN-γ IL-6, IL-8,IL-12, IL-17, and IL-18 in patients with active psoriasis and correlation with disease severity. Mediators of Inflammation. 2005 (5), 273-279 (2005).
  54. Grossman, R. M., et al. Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proceedings of the National Academy of Sciences of the United States of America. 86 (16), 6367-6371 (1989).
  55. Nair, R. P., et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways. Nature Genetics. 41 (2), 199-204 (2009).
  56. Boukamp, P., et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. The Journal of Cell Biology. 106 (3), 761-771 (1988).
  57. Deyrieux, A. F., Wilson, V. G. In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line. Cytotechnology. 54 (2), 77-83 (2007).
  58. Micallef, L., et al. Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes. Experimental Dermatology. 18 (2), 143-151 (2009).
  59. Wilson, V. G. Epidermal Cells. , Springer. 33-41 (2013).
  60. Ding, J., et al. Gene expression in skin and lymphoblastoid cells: Refined statistical method reveals extensive overlap in cis-eQTL signals. American Journal of Human Genetics. 87 (6), 779-789 (2010).
  61. Res, P. C., et al. Overrepresentation of IL-17A and IL-22 producing CD8 T cells in lesional skin suggests their involvement in the pathogenesis of psoriasis. PLoS One. 5 (11), (2010).
  62. Groves, R. W., Mizutani, H., Kieffer, J. D., Kupper, T. S. Inflammatory skin disease in transgenic mice that express high levels of interleukin 1 alpha in basal epidermis. Proceedings of the National Academy of Sciences. 92 (25), 11874-11878 (1995).
  63. Chan, J. R., et al. IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis. The Journal of Experimental Medicine. 203 (12), 2577-2587 (2006).
  64. Zheng, Y., et al. Interleukin-22, a TH 17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature. 445 (7128), 648-651 (2007).
  65. Ma, H. -L., et al. IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation. The Journal of Clinical Investigation. 118 (2), 597-607 (2008).
  66. Lowes, M. A., Russell, C. B., Martin, D. A., Towne, J. E., Krueger, J. G. The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses. Trends Immunol. 34 (4), 174-181 (2013).
  67. Banno, T., Gazel, A., Blumenberg, M. Effects of tumor necrosis factor-α (TNFα) in epidermal keratinocytes revealed using global transcriptional profiling. Journal of Biological Chemistry. 279 (31), 32633-32642 (2004).
  68. Boniface, K., et al. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. The Journal of Immunology. 174 (6), 3695-3702 (2005).
  69. Boniface, K., et al. Oncostatin M secreted by skin infiltrating T lymphocytes is a potent keratinocyte activator involved in skin inflammation. The Journal of Immunology. 178 (7), 4615-4622 (2007).
  70. Terui, T. Role of neutrophils in induction of acute inflammation in T-cell-mediated immune dermatosis, psoriasis: A neutrophil-associated inflammation-boosting loop. Experimental Dermatology. 9, (2000).
  71. Chiang, C. -C., Cheng, W. -J., Korinek, M., Lin, C. -Y., Hwang, T. -L. Neutrophils in psoriasis. Frontiers in Immunology. 10, 2376(2019).
  72. Kennedy-Crispin, M., et al. Human keratinocytes' response to injury upregulates CCL20 and other genes linking innate and adaptive immunity. The Journal of Investigative Dermatology. 132 (1), 105-113 (2012).
  73. Morizane, S., Gallo, R. L. Antimicrobial peptides in the pathogenesis of psoriasis. The Journal of Dermatology. 39 (3), 225-230 (2012).
  74. Vandal, K., et al. Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide. The Journal of Immunology. 171 (5), 2602-2609 (2003).
  75. Ryckman, C., Vandal, K., Rouleau, P., Talbot, M., Tessier, P. A. Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion. The Journal of Immunology. 170 (6), 3233-3242 (2003).
  76. Nukui, T., et al. S100A8/A9, a key mediator for positive feedback growth stimulation of normal human keratinocytes. Journal of Cellular Biochemistry. 104 (2), 453-464 (2008).
  77. Yang, D., et al. β-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science. 286 (5439), 525-528 (1999).
  78. Hsu, K., et al. Anti-infective protective properties of S100 calgranulins. Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Inflammatory and Anti-Allergy Agents). 8 (4), 290-305 (2009).
  79. Nestle, F. O., Kaplan, D. H., Barker, J. Psoriasis. The New England journal of medicine. 361 (5), 496-509 (2009).
  80. Tsoi, L. C., et al. Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity. Nature Genetics. 44 (12), 1341(2012).
  81. Swindell, W. R., et al. RNA-seq identifies a diminished differentiation gene signature in primary monolayer keratinocytes grown from lesional and uninvolved psoriatic skin. Scientific Reports. 7 (1), 1-13 (2017).
  82. Candi, E., Schmidt, R., Melino, G. The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol. 10 (4), 328-340 (2005).
  83. Frost, P., Weinstein, G. D., Van Scott, E. J. The ichthyosiform dermatoses II. Journal of Investigative Dermatology. 47, 561-567 (1966).
  84. Reichelt, J., Furstenberger, G., Magin, T. M. Loss of keratin 10 leads to mitogen-activated protein kinase (MAPK) activation, increased keratinocyte turnover, and decreased tumor formation in mice. Journal of Investigative Dermatology. 123 (5), 973-981 (2004).
  85. Hu, Z., et al. Loss-of-function mutations in filaggrin gene associate with psoriasis vulgaris in Chinese population. Human Genetics. 131 (7), 1269-1274 (2012).
  86. Giardina, E., et al. Characterization of the loricrin (LOR) gene as a positional candidate for the PSORS4 psoriasis susceptibility locus. Annals of Human Genetics. 68 (6), 639-645 (2004).
  87. Suga, H., et al. Skin barrier dysfunction and low antimicrobial peptide expression in cutaneous T-cell lymphoma. Clinical Cancer Research. 20 (16), 4339-4348 (2014).
  88. Iotzova-Weiss, G., et al. S100A8/A9 stimulates keratinocyte proliferation in the development of squamous cell carcinoma of the skin via the receptor for advanced glycation-end products. PLoS One. 10 (3), (2015).
  89. Seo, M. -D., Kang, T. J., Lee, C. H., Lee, A. -Y., Noh, M. HaCaT keratinocytes and primary epidermal keratinocytes have different transcriptional profiles of cornified envelope-associated genes to T helper cell cytokines. Biomolecules & Therapeutics. 20 (2), 171-176 (2012).
  90. Soboleva, A. G., et al. Genetically predetermined limitation in the use of HaCaT cells that affects their ability to serve as an experimental model of psoriasis. Genetika. 50 (10), 1222-1231 (2014).
  91. Wang, Z., et al. Systematic screening and identification of novel psoriasis-specific genes from the transcriptome of psoriasis-like keratinocytes. Molecular Medicine Reports. 19 (3), 1529-1542 (2019).
  92. Inoue, K., et al. Mechanism underlying ATP release in human epidermal keratinocytes. The Journal of Investigative Dermatology. 134 (5), 1465-1468 (2014).
  93. Harden, J. L., et al. CARD14 expression in dermal endothelial cells in psoriasis. PLoS One. 9 (11), 111255(2014).
  94. Krueger, J. G., et al. IL-17A is essential for cell activation and inflammatory gene circuits in subjects with psoriasis. The Journal of Allergy and Clinical Immunology. 130 (1), (2012).
  95. Hegyi, Z., et al. Vitamin D analog calcipotriol suppresses the Th17 cytokine-induced proinflammatory S100 "alarmins" psoriasin (S100A7) and koebnerisin (S100A15) in psoriasis. The Journal of Investigative Dermatology. 132 (5), 1416-1424 (2012).
  96. Schonthaler, H. B., et al. S100A8-S100A9 protein complex mediates psoriasis by regulating the expression of complement factor C3. Immunity. 39 (6), 1171-1181 (2013).
  97. Wilsmann-Theis, D., et al. Among the S100 proteins, S100A12 is the most significant marker for psoriasis disease activity. Journal of the European Academy of Dermatology and Venereology : JEADV. 30 (7), 1165-1170 (2016).
  98. Leuner, K., et al. Reduced TRPC channel expression in psoriatic keratinocytes is associated with impaired differentiation and enhanced proliferation. PLoS One. 6 (2), 14716(2011).
  99. Gao, L., et al. Ozone therapy promotes the differentiation of basal keratinocytes via increasing Tp63-mediated transcription of KRT10 to improve psoriasis. Journal of Cellular and Molecular Medicine. 24 (8), 4819-4829 (2020).
  100. Kim, B. E., et al. TNF-α downregulates filaggrin and loricrin through c-Jun N-terminal kinase: role for TNF-α antagonists to improve skin barrier. The Journal of Investigative Dermatology. 131 (6), 1272-1279 (2011).

访问受限。请登录或开始试用以查看此内容。

重印与许可

标签

视频即将推出
联系我们
向图书馆推荐

隐私

使用条款

政策

版权所有 © 2026 MyJoVE Corporation。保留所有权利。