【作者】 张洁；

【导师】 李宁丽；

【作者基本信息】 上海交通大学 ， 免疫学， 2016， 博士

【摘要】 银屑病,俗称“牛皮癣”,是一种以鳞屑性皮损为主要特征的常见慢性炎症性皮肤疾病,主要包括寻常型、红皮病型和脓疱型三种类型。目前研究发现,银屑病发病机制一方面由于角质形成细胞(Keratinocytes,KC)过度活化而引起表皮过度增生、角化不全,另一方面由于多种炎症细胞局部大量浸润,最终导致皮肤表面斑块状区域鳞屑脱落以及皮肤局部慢性炎症,但具体发病机理还有待进一步研究。CCN1,又称富含半胱氨酸蛋白61(Cysteine-rich angiogenic inducer 61,Cyr61)。是一种分泌型基质蛋白。目前已经公认CCN1作为一种新促炎症因子,参与多种炎症及自身免疫病发生。2015年,我们团队在国际上首次报道CCN1能通过促进KC过度活化、增殖加剧银屑病皮肤损伤。最近,我们还发现CCN1能促进KC产生IL-6、IL-8、IL-1β而介导银屑病发生,提示CCN1可能通过多种途径参与银屑病发生。但CCN1在银屑病疾病时相中扮演什么样的角色,是在疾病发生起始作为始动因素出现发挥对其他因子的促进作用?还是出现在疾病的后期起到炎症的维持及扩大作用还不清楚。近年研究表明,在银屑病病人皮损部位IL-36α、IL-36β、IL-36γ均高表达;IL-36α-/-小鼠咪喹莫特造模后皮损明显减轻;过表达IL-36α小鼠自发产生银屑病样皮损;IL-36可协同IL-1α加剧银屑病局部炎症。提示IL-36在银屑病的发生发展中十分重要。因此,寻找IL-36上游的调控因素对于有效抑制IL-36引起的炎症反应具有重要的研究意义及应用前景。在本论文中,我们首先建立了IMQ/IL-23诱导的银屑病样小鼠模型,并取不同时间点皮损分析炎症因子及CCN1的表达格局。结果表明CCN1和炎症因子TNF-α、IL-17、IL-23在造模第2天就迅速升高并达到峰值,提示CCN1和TNF-α、IL-17、IL-23均在造模早期表达,可能参与银屑病发生的起始。反之,IL-36α、IL-36β和IL-36γ在造模第7天开始升高并达到峰值,提示IL-36α、IL-36β和IL-36γ在造模晚期表达,可能参与银屑病的维持和复发。进而建立了我们的假设:“在银屑病疾病发生发展中CCN1可能处于IL-36的上游,并通过对IL-36的调控参与银屑病的发病”。接下来,我们在体外培养的KC细胞及角质形成细胞系(HaCaT)中研究CCN1对IL-36的体外调控作用。结果表明,外加CCN1蛋白或者转染CCN1表达质粒增加内源性CCN1表达均能选择性促进IL-36α和IL-36γ表达升高,而干扰CCN1内源性表达可有效抑制IL-36α和IL-36γ表达。通过与IL-36已知上游调控因素TNF-α、IL-17、IL-22对比研究,我们发现CCN1对IL-36的调控作用并不依赖TNF-α、IL-22,同时TNF-α、IL-17、IL-22调控IL-36表达的作用也不依赖CCN1,提示CCN1调控IL-36作用是区别于TNF-α、IL-17、IL-22调控途径的平行途径。进而,我们在IMQ诱导的银屑病样小鼠模型中,采用抗CCN1特异性单克隆抗体体内阻断CCN1表达,分析其对高表达的IL-36的作用。我们发现体内干扰CCN1表达能有效下调IL-36α和IL-36γ表达,从而缓解局部炎症和皮损症状。说明CCN1作为IL-36上游通过促进其高表达参与银屑病发生,提示CCN1可以被视为银屑病治疗的潜在靶点。此外,通过采用信号通路抑制剂及启动子结合位点预测,我们进一步分析了CCN1调控IL-36的分子机制。结果表明,CCN1选择性上调IL-36α和IL-36γ表达,是通过特异性结合KC细胞表面整合素受体α6β1,启动下游PI3K-AKT-NF-ΚB及ERK-AP1两条信号途径,使p65和ERK的磷酸化增加并结合IL-36α和IL-36γ启动子区使其表达增加。总之,我们的研究发现:CCN1不仅能够通过促进银屑病KC的过度增生参与银屑病的发生,更能够通过与炎症因子IL-17、IL-22和TNF-α平行的途径调控IL-36α和IL-36γ的表达来参与银屑病的发病。表明CCN1可通过多种机制参与银屑病KC与炎症微环境的相互作用网络,促进了银屑病病理过程的发生发展。综上,本研究利用IMQ/IL-23诱导的银屑病样小鼠模型、体外原代培养的KC及HaCaT角质形成细胞系,在体内和体外全面分析了CCN1介导IL-36表达的新机制和新思路,证实了CCN1不仅能够通过参与KC过度活化增殖参与疾病,而且能够通过促进IL-36表达扩大炎性微环境,广泛参与银屑病发病。同时,体内阻断CCN1功能可以有效抑制IL-36及炎症因子的表达,从而缓解小鼠皮肤损伤和炎症反应,提示我们,CCN1可能是银屑病临床治疗的潜在靶标,为银屑病的机理研究和临床治疗提供了有力的新证据。更多还原

【Abstract】 Psoriasis is a popular chronic inflammatory skin disease,which includs ordinary Psoriasis,erythrodermic Psoriasis and pustular Psoriasis characterized by red,scaly and well-demarcated skin lesions formed by the hyper-proliferation of epidermal keratinocytes（KC）.The pathogenesis includes epidermal keratinocytes hyper-proliferation which is caused by excessive activation of hyperplasia,parakeratosis and inflammatory cell infiltration leading to patchy skin surface area scales fall off as well as local chronic inflammation of the skin.In recent years,numerous studies show that Psoriasis symptoms（such as keratinocyte hyperplasia,neovascularization,inflammation,etc.）can be attributed to a large number of local infiltration of inflammatory cytokines.CCN1,also known as Cysteine-rich protein 61（Cysteine-rich angiogenic inducer61,Cyr61）,is a secreted matrixcelluar protein.CCN1 is now recognized as a new pro-inflammatory cytokine,which is involved in a variety of inflammatory and autoimmune diseases.In 2015,we reported that CCN1 promots excessive keratinocytes activation,proliferation in aggravating psoriasis skin lesions.Recently,we found CCN1 can promote keratinocytes to produce IL-6,IL-8 and IL-1β.CCN1may be involved in psoriasis via a variety of pathways.But there are still a lot of problem yet and need to be solved.What is the expressive phase of CCN1 in psoriasis.What role does it play?Is CCN1 induced in the initiation of the disease or expresss high in the late phase of Psoriasis.Recent studies showed that in patients with psoriatic skin lesions,IL-36α,IL-36β,IL-36γwere highly expressed.In addition,significantly reduced psoriasis-like symptoms were observed in imiquimod treated IL-36α^-/-mice.While overexpression of IL-36αin mice spontaneously generates psoriasis-like lesions.It has been reported that IL-36 synergistically assists IL-1αto increase local inflammation in psoriasis.All of these studies illustrate that IL-36 is important in the development of psoriasis.Therefore,it is significant and applicable to find the factors regulating IL-36 to block the disease from the orgin.In this research,we first established the IMQ/IL-23-induced psoriasis-like mouse model,then collected all skin samples at different time points to analyze the expression pattern of inflammatory factors and CCN1.The results showed that CCN1and inflammatory cytokines TNF-α,IL-17,IL-23 quickly increased from the first two days of modeling and reached the peak,which suggests that CCN1,TNF-α,IL-17,and IL-23 may be involved in the initial occurrence of psoriasis.Conversely,IL-36α,IL-36βand IL-36γare significantly induced on the seventh day from the beginning of the modeling,which suggests that IL-36α,IL-36βand IL-36γmay be involved in psoriasis maintenance and relapse.Thereby we established our hypothesis:"CCN1may be involved in the pathogenesis of psoriasis via regulating IL-36 expression."Next,we studied the regulation between CCN1 and IL-36 in cultured KC and HaCaT cell line in vitro.The results showed that both CCN1 protein extra-added or PCDH-CCN1 expression plasmid transfection are able to selectively promote IL-36αand IL-36γexpression.On the other hand,IL-36αand IL-36γexpression were selectively inhibited upon CCN1 interference.In addition,it is well known that TNF-α,IL-17,IL-22 can induce IL-36 expression.So what is the relationship between these already known cytokines and CCN1 in regulating IL-36 production?Our data showed that CCN1,which is not involved in the regulatory effect of TNF-α,IL-17and IL-22 on IL-36 expression,can induces IL-36 expression directly without the involvement of TNF-αand IL-22.So we concluded that CCN1 regulates IL-36 in a parallel regulatory pathway with TNF-α,IL-17,IL-22 regulation.Further,we established IMQ-induced psoriasis-like model and treated the model mice by specific anti-CCN1 monoclonal antibodies to block the expression of CCN1and analyzed the expression of IL-36.We found that CCN1 expression inhibition in vivo can selectively reduce IL-36αand IL-36γexpression resulting in a relieved symptom.In addition,using signal pathway inhibitor and predicting promoter binding sites,we further analyzed the molecular mechanisms of CCN1 regulating IL-36.The results showed that,CCN1 selectively upregulated IL-36αand IL-36γexpression through the specific combination of KC cell surface integrin receptorsα6β1,signaling through PI3K-AKT-NF-κB and ERK-AP1 pathways,then increased p65 and ERK phosphorylation to the nucleus and binding to IL-36αand IL-36γpromoter to start the expression.In conclusion,our study found that:CCN1 not only participates in promoting psoriasis KC hyper-proliferation,but also play an important role in expanding inflammation during disease progression,such as regulation of IL-1β,IL-36αand IL-36γto participate in the expansion of local inflammation in psoriasis as an up-stream origin.Therefore,CCN1,from different aspects,became involved in the network of psoriatic KC and inflammatory microenvironment promoting the development of pathological processes of psoriasis.Moreover,our researches provided the experimental and theoretical basis of CCN1 for the clinical treatment of psoriasis as a new target.In summary,we found CCN1 can regulate IL-36 expression via AKT-NF-κB/ERK-MAPK pathways to participate in Psoriasis in vitro and in vivo to expand the inflammatory microenvironment which plays an important role in the pathogenesis of psoriasis.Also,blocking CCN1 function significantly inhibit skin damage and inflammation,suggesting that CCN1 may be a potential target for clinical treatment of psoriasis and psoriasis research,and provided strong new evidence in the mechanism of clinical treatment.更多还原