【作者】 吕景晶；

【导师】 白彦萍；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 银屑病俗称“牛皮癣”,中医称之为“白疙”,目前被认为是一种以红斑鳞屑为主要临床表现的慢性、炎症性皮肤病,通常侵犯皮肤和关节,且易复发。近年来发病率有逐渐增高的趋势,银屑病的确切病因仍有待研究,多数专家认为银屑病是由遗传因素和环境因素等多种因素相互交织作用所导致。其整个发病过程受到细胞因子网络与多种信号通路的调控。Notch信号通路由Notch受体、Notch配体和CSL DNA结合蛋白三部分组成。广泛分布于造血干细胞,胚胎干细胞,淋巴细胞,血管内皮细胞等多种细胞表面。研究结果表明,Notch信号在银屑病的真皮处于活化状态,可通过影响T细胞的发育、活化及调节细胞因子的分泌参与银屑病的发生。有研究显示在包括角质形成细胞等多种表皮细胞的表面,Notch及其配体均有大量表达,且特异性的Notch信号活化在维持皮肤细胞自身更新及分化过程中起重要作用。异常的Notch信号可能有助于揭示银屑病等皮肤疾病的发病机制。目前国内外药物干预Notch信号通路治疗银屑病的研究仍处于空白阶段,作用机理研究更为匮乏。因此,从免疫调节角度进行调控Notch信号通路治疗银屑病的药物研究十分必要,且具备广泛的探索空间。血热型银屑病相当于银屑病的初期(急性期)是临床表现最典型且多见的类型,祛银颗粒是导师白彦萍教授的自拟方药,以清热凉血解毒中药为主要成分,其良好的临床疗效已通过前期的临床及动物模型研究得到证实。但其具体作用机制和作用靶点尚不清楚。本研究旨在从分子生物学角度探讨Notch信号传导分子是否在血热型银屑病患者中发挥一定的作用。了解各传导分子的活跃情况将有助于支持后续清热凉血方药对Notch信号通路调控的相关研究。目的：揭示血热型银屑病患者单个核细胞中Notch信号分子(Notch1-4、Delta1、 Delta3-4、Jaggedl-2)的表达情况,并对银屑病患者的病情严重程度与Notch信号分子的表达关系做出客观评价。方法：收集纳入血热型银屑病患者,进行PAS I评分并采集患者外周血,提取出单个核细胞。以健康人为参照,分别采用实时荧光定量PCR法检测27例血热型患者和27例健康人的Notch信号分子mRNA表达水平；运用流式细胞术对6例血热型银屑病患者和5例健康人的Notch信号分子平均荧光强度的表达进行检测；采用蛋白免疫印记法(Western Blot)检测血热型银屑病30例和健康人30例的Notch信号通路的相关蛋白表达。结果：(1)血热型银屑病患者单个核细胞中Notch2的mRNA水平较健康人显著升高,两者间差异有统计学意义,P=0.036(P<0.05),其表达与PASI评分呈正相关趋势。(2)血热型银屑病患者单个核细胞中Notch3的mRNA水平较健康人显著降低,两者间有显著性差异,P=0.008(P<0.01),未发现其表达与PASI评分呈正相关趋势。(3) Notch1、Notch4、Jagged1-2、Delta1、Delta3-4,在血热型银屑病患者单个核细胞中的mRNA水平较健康人无明显差异,两者间无统计学意义(P>0.05)。(4)血热型银屑病患者组的Notch2平均荧光强度(MFI)低于健康人对照组的Notch2MFI表达水平,两者比较有显著差异(P<0.01),其与PASI评分无明显相关性。(5)血热型银屑病患者组的Notch1、Noth3平均荧光强度(MFI)与健康人对照组的Notch1、Noth3MFI两者比较无统计学意义(P>0.05)。(6) Western Blot显示健康人组与血热型患者组比较,血热型患者组Delta4蛋白表达明显上调,有显著统计学差异(P<0.01)。其与PASI评分无明显相关性。(7) Western Blot显示健康人组与血热型患者组比较,Notch1、Deltal蛋白表达无明显差异,两者间无统计学意义(P>0.05)。结论：Notch2在mRNA和蛋白表达方面较健康人均有所升高,Delta4的蛋白表达较健康人升高。其中Notch2mRNA与表达与银屑病的严重程度呈正相关；Notch3mRNA表达较健康人有所下降,但其与银屑病的严重程度无明显相关性。Notch信号通路参与了血热型银屑病的发生、发展等调节过程。更多还原

【Abstract】 Background:Psoriasis, also known as "Niu Pi Xuan", or called "Bai Bi" in Traditional Chinese Medicine (TCM), currently has been regarded as a kind of chronic skin disease of inflammation, relatively appeared in clinic with erythema scale as its main clinic symptom. It often occurs on skin and joints and easily relapses. The incidence of psoriasis is gradually increasing in recent years; however, the specific causes of psoriasis are still unclear. Most experts believe psoriasis is a genetic disease related to multiple genes, which is caused and affected by the interaction of multiple factors, such as genetic and environmental factors. During the whole progress of disease, it is influenced and regulated by multiple complex cytokine networks and signaling pathways.Notch signaling pathway consists of Notch acceptors, Notch ligands and CSL DNA-binding protein. It is widely distributed in the surface of multiple cells, such as hematopoietic stem cells, embryonic stem cells, lymphocytes, and vascular endothelial cells. Some studies show that Notch signal is activated in the dermis, and it can promote the onset of psoriasis by influencing the development and activation of T cells and regulating the secretion of cytokine. Some studies indicate that there is high expression of Notch and its ligand in the surface of keratinocyte and other epidermal cells and the specific activation of Notch signaling plays an important role for maintaining the skin cells’ renewal and differentiation. Therefore, abnormal Notch signals possibly help to reveal the pathogenesis of psoriasis and other skin diseases.To date, there is no domestic or overseas research exploring certain medications for the treatment of psoriasis by regulating the Notch signaling pathway, and research on the mechanism of the treatment is even rarer. Therefore, it is extremely necessary to conduct an intervention study from immune regulation perspective to treat psoriasis by regulating Notch signaling pathway. Blood-heat psoriasis, equivalent to the initial stage or acute stage of psoriasis, is the most typical and common type of psoriasis in clinical practice. Qu Yin Granules is a self-prescribed prescription by Doctoral Supervisor and Professor Bai Yanping. Its essential components consist of Chinese herbal medicines with functions of clearing heat, cooling blood and resolving toxins. We has confirmed its beneficial effects on psoriasis by previous clinical studies and animal experiments. However, the specific mechanism and the action target are still unclear. This study aims at probing whether the Notch signal transduction molecules play a role or not in blood-heat psoriasis from the perspective of molecular biology. Understanding of the active situation of the transduction molecules will support future research on the regulation of Notch signaling pathway by clearing-heat and cooling-blood medications, which will lay a foundation for revealing the whole mechanism of the treatment.Purposes:The purposes of this research are to reveal the expression of Notch signaling molecules (Notch1-4, Deltl, Delt3-4and Jagged1-2) in mononuclear cells of blood-heat psoriasis patients, and to objectively evaluate the relationship between the psoriasis severity and the expression of Notch signaling molecules, and to explore target protein molecules for the regulation of Notch signaling pathway.Methods:27blood-heat psoriasis patients confirmed under the guidance of TCM theory were allocated in the experimental group and graded with PASI scores, with27health participants as control. We collected the peripheral blood samples of all participants to extract their mononuclear cells. Real-time fluorescent quantitation PCR method was used to detect the expression levels of mRNA of Notch signaling molecules of all participants. Flow cytometry was used to record expressions of the mean fluorescence intensity (MFI) of Notch signaling molecules in6blood-heat psoriasis patients and5healthy participants. Western Blotting was used to detect the expression of proteins related to Notch signaling molecules of30participants in experimental group and30healthy participants in control group.Results:(1)Compared with healthy participants, blood-heat psoriasis patients had a marked increase of mRNA level of Notch2in mononuclear cells. There was a significant difference between groups, P=0.036(P＜0.05). The expression was positively correlated with PASI scores.(2)Compared with healthy participants, blood-heat psoriasis patients had an obvious decrease of mRNA level of Notch3in mononuclear cells. There was a significant difference between groups, P=0.008(P＜0.01). The positive correlation between the expression and PASI scores was not detected.(3)Compared with healthy participants, there were no specific changes of Notchl, Notch4, Jaggedl-2, Deltal and Delta3-4in blood-heat psoriasis patients. There was no significant difference between them.(P＞0.05)(4)There was significant statistical difference (P＜0.01) for Notch2MFI between experimental group and healthy group. The PASI correlation analysis showed that the Notch2’s flow MFI expression was independent of the psoriasis severity.(5) There was no statistical significance between the MFI of Notchl and Notch3of experimental group and Notchl MFI of healthy group.(6) Western Blotting showed that there was an obvious increase of Delta4protein expression, with significant statistical difference (P＜0.01). The PASI correlation analysis showed that Delta4protein was independent of the psoriasis severity.(7)Western Blotting showed that there was no difference (P＞0.05) for Notchl or Delta4protein expression between experimental group and healthy group.Conclusions:The mRNA and protein expressions of Notch2of blood-heat psoriasis patients were higher than healthy participants, and the higher of mRNA level of Notch2, the severer of psoriasis, which indicates the direct relation between psoriasis severity and the activity level of Notch2. As Notch signaling molecule ligand, Delta4has directly participated in the course of blood-heat psoriasis. Though psoriasis participants had a decrease of Notch3mRNA expression, compared to healthy people, it cannot be used as diagnostic standards for psoriasis. Notch signaling pathway directly has participated in the onset and development of blood-heat psoriasis.更多还原