银屑病中PD-1/TCR的阴阳平衡免疫学机制及芩珠凉血方的调控作用研究

The skin epithelium and the connective tissue of the innate and adaptive immune system is an important cause of abnormal feedback mechanism in the pathogenesis of psoriasis. Previous studies found that Qinzhuliangxue decoction treatment reduced the blood heat syndrome and repressed the expressions of Th1 type cytokines IL-2 and IFN-γ in patients with psoriasis.of . Here we will test our hypothesis that psoriasis is a disease that imbalance between sensors/balances (such PD-1, YIN) and activators (such as TCR, YANG) in T cell activation. Qinzhuliangxue decoction treatment repressed activators such as TCR and activate balancers, such as PD-1 to rebalance the T cells in skin to treat psoriasis. Specifically, in psoriasis mouse models and cellular studies will be performed to test our hypothesis. Some translational research will also be performed. In mouse models studies, imiquimod-induced mouse model of psoriasis will be used to identify the balance between PD-1 and TCR in psoriasis development and the treatment effects of Qinzhuliangxue decoction. In cellular studies, conditional medium will be collected from T cell cultures and will be used to stimulate keratinocytes to identify the role of cytokine in PD-L1/2 activation and the interactions between T cells and keratinocytes. We will also collect skin samples from Qinzhuliangxue-treated patients toi monitor the changes of PD-1 and TCR in psoriasis progression and recovery and the effects of Qinzhuliangxue in PD-1/TCR balance. This project will disclose some new immunity mechanisms in psoriasis development and determine the immunology mechanisms in traditional Chinese medicine in the treatment of psoriasis. Our long-term goal is to develop new therapeutic strategies for patients with psoriasis.

皮肤上皮细胞与结缔组织的固有免疫与适应性免疫系统的异常反馈是导致银屑病发病的重要机制。前期研究发现，芩珠凉血方可降低银屑病血热证患者的Th1 型细胞因子 IL-2、IFN-γ ，起到调节银屑病血热证 T 细胞活化的作用。我们推测银屑病是皮肤T细胞启动的“感受子”和“平衡子”(PD-1相关蛋白，阴)与“启动子”（T细胞受体（TCR）相关蛋白，阳）的失衡，芩珠凉血方可以通过启动PD-1通路、抑制TCR通路，平衡阴阳，以治疗银屑病。本项目通从临床、动物模型和细胞生物学水平上研究芩珠凉血方对银屑病关键基因表达及重要信号分子效应的调控，以诠释银屑病发生发展的免疫学机制，并从PD-1/PD-L与Th17/TCR之间阴阳平衡的效应机制探求芩珠凉血方治疗“血热证”银屑病的细胞生物学基础，对确定中医药治疗银屑病的靶点以及中医临床客观诊疗具有积极意义。

基于前期研究证实“清热凉血法代表方-芩珠凉血方”能有效治疗“血热证”银屑病患者，且可降低银屑病血热证患者的Th1 型细胞因子 IL-2、IFN-γ，起到调节银屑病血热证T细胞活化作用。近期研究表明PD-1与其配体结合可以抑制银屑病发生发展的中心环节T淋巴细胞的活化和增殖。在此基础上我们进一步探讨银屑病中PD-1/TCR的阴阳平衡免疫学机制及芩珠凉血方的调控作用。临床研究：我们以寻常型银屑病“血热证”患者、正常人（健康对照）为研究对象，通过PCR-Sanger测序分析对银屑病“血热证”患者和健康对照进行六个PDCD1 SNP（PD1.1，PD1.3，PD1.4，PD1.5，PD1.6和PD1 .9）测序；通过Real-time PCR 技术比较 PD-1、PD-L、IL-17A等基因 mRNA 在各组间的表达水平；运用免疫组化法进一步观察 PD-1、PD-L、TCR在不同组别的分布特点。研究结果提示： PD1.6在基因型和等位基因频率分布上与银屑病显著相关。 PD1.6的G等位基因增加了发生银屑病的风险。免疫组化的结果从蛋白层面提示银屑病“血热证”患者局部皮损中的TCRβ表达信号增强，导致PD-1的抑制效应减弱，使得PD-1与PD-L2的结合降低，进而不能有效抑制银屑病的关键致病细胞因子IL-17A、I的表达，从而导致银屑病的发生发展。Real-time PCR的相对定量结果从基因层面肯定了免疫组化的结果。通过芩珠凉血方治疗血热证银屑病，我们发现其疗效确切。银屑病血热证患者服用芩珠凉血方起效后，使得血热证患者局部皮损中的TCRβ表达信号降低，导致PD-1的抑制效应增强，从而提高患者皮损局部的PD-1及PD-L2的表达，使得两者结合量增大，能有效地抑制T细胞的活化，从而降低银屑病发病关键的细胞因子IL-17A的皮损局部表达而起到有效治疗银屑病的作用。我们目前的实验结果很好地诠释芩珠凉血方可以通过启动PD-1 通路，抑制TCR通路，平衡阴阳，治疗银屑病的假说。本研究阐释芩珠凉血方治疗银屑病的细胞生物学基础，对确定中医药治疗银屑病的靶点以及中医临床客观诊疗提供新的思路，具有重要的理论意义及潜在应用价值。

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