基于免疫微环境对角质形成细胞影响研究银屑病"血热证"细胞生物学机制

According to the blood heat syndrome of Psoriasis Vulgaris caused by Blood and Heat in Traditional Chinese medicine, and the epidermal keratinocytes abnormal differentiation caused by immune microenvironment changes in modern medicine, both the clinical signs of erythema, scaling, itching. Our group on the basis of previous work,Combined modern immunology and cell biology progress, Put forward the Scientific hypothesis: keratinocyte cell immune changes in the microenvironment is one of the cause of psoriasis blood heat syndrome key material basis. This project uses a combination of in vivo and in vitro experiments, integrated use of the Solexa high-throughput sequencing technology, Realtime-PCR, immunohistochemistry, Western blot technology, systematic observed psoriasis Vulgaris "blood heat syndrome" in patients with normal tissue gene and protein expression differences. Through recipe measurements Syndrome type we screened of the drug of clear away heat and blood sensitivity gene, confirmed and evidence to the contrary of blood heat syndrome symbol of differentially expressed genes.Normal and the patients of psoriatic blood heat syndrome Serum culture of keratinocytes study using siRNA technology, resulting in a related gene silencing, and thus depth study of the blood heat syndrome critical gene function，in order to explain the Cell Biology mechanism of psoriasis blood heat syndrome, to provide a basis to explore the psoriatic blood heat syndrome material basis.

根据中医"血分热甚"所引起的银屑病血热证，和免疫微环境改变导致的表皮角质形成细胞异常分化，两者共同存在"红斑、鳞屑、瘙痒"的临床体征，课题组在前期工作基础上，结合现代免疫学、细胞生物学进展，提出"免疫微环境改变对角质形成细胞的作用是导致银屑病血热证的关键物质基础之一"的科学假说。本项目采用在体与离体实验相结合，运用Solexa高通量测序技术，Realtime-PCR，免疫组化，Western blot等技术，系统观察银屑病"血热证"患者与正常人组织中相关基因、蛋白表达差异，通过以方测证筛选银屑病"血热证"清热凉血药物敏感基因，印证和反证血热证的标志性差异基因。并以正常人和银屑病"血热证"患者血清培养的角质形成细胞为研究对象，采用siRNA干扰技术，造成相关基因沉默,进而深入研究"血热证"关键基因功能，从而阐释银屑病"血热证"的细胞生物学机制，为探求银屑病"血热证"物质基础提供依据。

根据中医“血分热甚”所引起的银屑病血热证和免疫微环境改变导致的表皮角质形成细胞异常分化，两者共同存在“红斑、鳞屑、瘙痒”的临床体征，课题组提出“免疫微环境改变对角质形成细胞的作用是导致银屑病血热证的关键物质基础之一”的科学假说。本项目采用在体与离体实验相结合，运用Solexa 高通量测序技术，Realtime-PCR，免疫组化，Western blot 等技术，系统观察银屑病“血热证”患者与正常人组织中相关基因、蛋白表达差异，通过以方测证筛选银屑病“血热证”清热凉血药物敏感基因，共涉及到Allograft rejection、Alzheimer's disease、Arachidonic acid metabolism、Complement and coagulation cascades、Drug metabolism、ECM-receptor interaction、Glycosaminoglycan degradation、Graft-versus-host disease、Huntington's disease、Linoleic acid metabolism、Lysosome、Metabolism of xenobiotics by cytochrome P450、Oxidative phosphorylation、Parkinson's disease、Prion diseases、Systemic lupus erythematosus和Type I diabetes mellitus等17条信号通路。根据课题组前期研究证实的Th17辅助T细胞及相关信号转导因子在寻常性银屑病血热证的免疫发病机制中占有重要作用。研究选取Allograft rejection和Graft-versus-host disease pathway重点关注，并最终聚焦PD-1/PD-Ls信号通路。经Realtime PCR、Western blot及免疫组化验证，清热凉血药物治疗寻常性银屑病血热证涉及PD-1，PD-L1，PD-L2，CTLA4，IL-17，IL-23等血热证标志性基因，清热凉血药物通过上调PD-1、PD-L1、PD-L2、CTLA4，下调IL-17、IL-23基因与蛋白的表达发挥治疗作用，并可检测上述指标，指导诊疗，明确预后。