从角质形成细胞K17/抗凋亡基因表达探讨银屑病血热证/血瘀证证型特点的分子机制

Psoriasis is a very common disease and hard to cure. The different of TCM patterns is the key point of TCM treatment and the good representation of psoriatic diversity characteristics. However, there is still uncertain on mechanism of TCM patterns differentiation and need to do more research. It has been previous confirmed that there is significantly different expression of keratin 17 in both TCM patterns (heat of blood and blood stasis). However, there is no significant difference in cytokines which inducing keratin 17 expression in both TCM patterns. We proposed that it exist different mechanisms in both of TCM patterns (heat of blood and blood stasis). The psoriatic differentially expressed genes in both of psoriatic TCM patterns (heat of blood and blood stasis) may have effect on the activity of anti-apoptotic signal channels association with K17, which then result in the expression of K17 and form the different TCM patterns. In this study, we detected the activity of anti-apoptotic signal channels association with K17 in two TCM patterns. By using Whole genome RNA sequencing, we detected the differentially anti-apoptotic expressed genes in both of psoriatic TCM patterns (heat of blood and blood stasis) and selected the anti-apoptotic encoding genes association with specific TCM patterns. And then using RNA interference method to silent the specific TCM pattern associating genes so as to investigate the apoptosis rate of keratinocytes and K17 expression. We try to deeply investigate the different molecular mechanisms in both of psoriatic TCM patterns (heat of blood and blood stasis) at genetics lever. It’s new insight to deepening understanding the essence of TCM patterns differentiation and to provide the objective evidence in precision therapy of psoriasis.

银屑病常见而难治，中医调治首当辨证。辨证分型也体现了银屑病的复杂多样，关于银屑病证型差异的分子机制研究还很少。前期研究表明银屑病血热证和血瘀证型存在银屑病角质形成细胞标志性蛋白—K17基因的差异表达；而诱导K17表达的细胞因子，证型间无差异。本研究提出调控血热证/血瘀证2种证型的分子机制不同，抗凋亡基因的差异表达，可以调控K17相关的信号通路的活性，影响角质形成细胞K17的表达，进而形成不同证型。本项目通过检测两证型K17相关信号通路的活性差异，并采用高通量的转录组测序技术（RNA-Seq)，筛选证型相关的抗凋亡差异表达基因，并在人HaCaT角质形成细胞中运用RNA干扰技术（RNAi）分别沉默证型相关基因，检测2种证型的细胞凋亡率、K17表达水平，以期从基因水平深入探寻银屑病血热证/血瘀证不同证型特点的分子机制，并从全新的视角认识中医证型差异的本质，为银屑病精准治疗提供更客观的证据。

银屑病（Psoriasis）是一种十分常见且顽固难治的皮肤病。迄今为止，银屑病仍不能完全性治愈。银屑病的临床表现其实并不完全相同，但西医多采用一贯制药物维持治疗，而且长期使用相同药物所产生的副作用和药物抵抗也为后续的治疗带来了很大的治疗难题。辨证论治是中医理论的精髓，辨证论治理论更好的体现了“同病异治”的中医理念，准确的中医分型是辨证论治的核心内容，只有辨证准确才可以发挥最佳的临床疗效。科学的阐明银屑病不同证型分子机制的差异，是提高银屑病辨证准确度的重要依据，也是中医药精准治疗的前提，但关于调控银屑病证型差异的分子机制研究还很少。为此，本研究应用现代的研究检测手段，通过转录组测序技术、细胞实验、组织学等方面深入研究银屑病血热证/血瘀证分子机制的差异性。研究发现血热证和血瘀证存在显著差异表达基因，这些基因与角质形成细胞增殖和分化相关。研究进一步证实银屑病血热证及血瘀证皮损处I型及II型角蛋白的组成及表达水平存在差异，与银屑病高度相关的KRT17/16/6A在银屑病血热证皮损中表达量显著高于银屑病血瘀证。研究还发现Nrf及其下游SPRR家族的表达水平在血热证/血瘀证中存在差异。Nrf通过调节不同SPRR的表达水平，影响STAT1/3通道的活性，进而调控银屑病角质形成细胞关键角蛋白（K6、K16和K17）的表达，最终形成血热证/血瘀证不同的临床表现。本研究从遗传和免疫机制的角度科学的认识了证型差异的本质，提高了银屑病辨证的准确度，也为银屑病中医精准治疗提供了更为客观的依据。