Pirt调节组胺H4受体介导的痒觉机理研究

Pirt is a novel membrane protein that is specifically expressed in the dorsal root ganglia. The Function of Pirt is not clear in the modulation of itch via histamine H4 recetpor. In this study, we will focus on how Pirt regulates itch which mediated by H4 recetpor. The hypothesis of the study is that pirt and H4 recetpor co-express on the dorsal root ganglia neuron; H4 recetpor is coupled to TRPV1. Pirt modulated itch which mediated by H4 recetpor via TRPV1. The multiple methods were employed that they are animal bebaivor, immunohistochemistry, real-time quantitative RT-PCR Western blot, cell culture，calcium imaging, and electrophysiology. We will find the difference of scratching which is induced by H4 agonist VUF-8430 the Pirt-/- and Pirt+/+ mice and the difference of pirt and H4 recetpor co-express on the DRG nueron in the Pirt-/- and Pirt+/+ mice. On the culture neurons of Pirt-/- and Pirt+/+mice, the whole cell current of H4 receptor were measured and analysised. On the HEK 293 and CHO cells co-expressed Pirt, TRPV1 and H4 receptor, the whole cell current were measured and analysised in oder to study how Pirt participate in the regulation of itch which mediated by H4 recetpor. We will try to demonstrate the relationship of Pirt, TRPV1 and H4 and confirm the role of Pirt in the modulation of itch which mediated by H4 recetpor. This study will help us to deepen understand of Pirt's function and enrich the knowledge of itch regulation.

Pirt是我们新发现的一种只表达在DRG神经元上的蛋白，是否调节组胺H4受体介导的痒觉尚不清晰，本项目将研究这一问题。研究提出理论假说：Pirt与H4受体在DRG神经元上共表达；TRPV1是H4受体下游耦联的离子通道；Pirt通过TRPV1调节H4受体介导的痒觉。研究采用行为学、免疫组化、钙成像、膜片钳记录等技术。比较Pirt-/-鼠与Pirt+/+鼠对h4受体激动剂诱发的搔痒行为差异；观察Pirt与H4受体在Pirt-/-鼠与Pirt+/+鼠DRG神经元上表达情况；分离培养Pirt-/-和Pirt+/+DRG神经元细胞，观察h4受体激动剂反应细胞数量的变化，观察Pirt如何调节激动H4受体的全细胞电流。在HEK293细胞或CHO细胞模型上,研究Pirt对H4受体介导的全细胞电流的影响。研究将明确Pirt调节H4受体介导的痒觉机制。增加对Pirt生理功能的了解，丰富痒觉调节机制的理论。

痒是一种不愉快的感觉，会引起人或者动物搔痒的反射或者抓痒的欲望。慢性瘙痒会给人带来沉重的负担，严重影响人们的生活质量。组胺H 4受体已经被确认它在外周的瘙痒中扮演重要角色。Pirt是特异性性表达在DRG神经元蛋白，它可以调节TRP通道电流。蛇床子素是一种天然香豆素，它可能参与调节组胺致痒。本项目通过行为学，钙成像，电生理等研究方法对H4受体的信号转导通路以及下游离子通道，pirt调节DRG神经元的钠电流，蛇床子素调节TRPV1的活性进行了研究。研究发现DRG神经元上的H4受体通过PLC通路偶联下游TRPV1通道。Pirt蛋白调节H4受体诱导的瘙痒行为，敲除pirt后，DRG神经元的动作电位幅度，阈值以及超射都会减弱。蛇床子素通过抑制TRPV1电流的方式抑制组胺引起的瘙痒。综上，TRPV1参与H4受体介导的瘙痒，蛇床子素可以通过调节TRPV1抑制组胺依赖的瘙痒。Pirt不是通过调节TRPV1方式调节H4受体介导的瘙痒，而是通过抑制钠电流。研究加深了我们对pirt蛋白的认识，同时阐明H4受体作用机制，为将来组胺依赖的瘙痒的药物开发提供新的靶点。