PPARδ在角质形成细胞及银屑病中的作用及其分子机制研究

发布时间：2018-04-22 10:32

本文选题：银屑病 + IMQ诱导的小鼠模型　；参考：《第二军医大学》2017年博士论文

【摘要】：银屑病是一种自发性的自身免疫性疾病,影响着约全球约2%的人口,在中国发病率稍低,但中国人口基数庞大,有着约500-800万的银屑病患者。由于银屑病目前还不能根治,且特别容易复发,给患者身心及家庭带来了严重的损害。咪喹莫特(imiquimod,IMQ)最初用于治疗人皮肤癌和外生殖器/肛周疣等疾病,后被研究者开发并用在小鼠皮肤上来诱导银屑病样的症状。由于该模型能很好的诱导出人银屑病的绝大部分症状,且模型简单易制备,迅速成为研究银屑病的一个重要模型。过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptors,PPARs)属于II型核受体超家族,可分为3种亚型(PPARα/γ/δ)。PPARδ在代谢综合征、动脉粥样硬化、脂代谢紊乱等疾病过程中发挥了重要的作用。前期的实验发现PPARδ在人银屑病皮损和IMQ诱导的小鼠银屑病皮肤中高表达,提示其可能在银屑病的发生发展中起到一定的作用。目前银屑病的治疗没有很好的办法,且不能彻底的治愈,本实验借助IMQ诱导的银屑病样小鼠以及人角质原代细胞进行一系列实验,为PPARδ在银屑病中的作用提供更为有力和直接的证据,以期为银屑病的治疗提供新的治疗靶点。第一部分PPARδ在银屑病皮损中的表达情况目的:为了考察PPARδ在人和小鼠银屑病皮损及正常皮肤中的表达情况,从而为PPARδ在银屑病中的作用提供依据。方法:取健康人皮肤和银屑病人皮损处皮肤,小鼠正常皮肤和IMQ刺激后的皮肤,进行免疫组织化学(immunohistochemistry,IHC)、实时荧光定量PCR(quantitative polymerase chain reaction,Q-PCR)和蛋白免疫印迹(Western Blot,WB)实验,考察PPARδ在正常皮肤和银屑病皮损中、表皮和真皮层中的表达差异。并进一步探讨了PPARδ在银屑病皮损部位的特异性表达。结果:人银屑病皮损和小鼠经IMQ刺激皮肤中的PPARδ表达较健康皮肤和正常小鼠皮肤均升高。通过IHC染色观察到,人皮损处的PPARδ主要表达于表皮上基底层。真皮、表皮的表达偏好来看,人及小鼠皮肤中PPARδ主要表达于表皮层,而真皮层中含量很少。PPARδ于健康人皮肤中表达很少,同时也很少表达于其它几种皮肤疾病的皮损中,表明了PPARδ在银屑病皮损部位表达的相对特异性。结论:PPARδ在人银屑病皮损处及IMQ诱导的小鼠银屑病皮损中有所升高,表明了PPARδ很可能在银屑病的发病中起到了很重要的作用。第二部分PPARδ对于原代角质细胞体外分泌细胞因子的作用目的:考察PPARδ在不同细胞炎症模型中所起的作用及其潜在机制。本部分我们一共借助了三类比较常规的与银屑病关系比较密切的细胞模型。(1)白介素(interleukin,IL)-17、IL-22和肿瘤坏死因子alpha(tumor necrosis factor,TNF-α)等细胞因子在银屑病的发展中起到重要的作用,我们探寻了PPARδ对于在这些细胞因子作用下角质细胞炎症反应的影响。(2)12-O-十四烷酰佛波醋酸酯-13(12-O-Tetradecanoylphorbol-13-acetate,TPA)在体内也常用于诱导小鼠的银屑病样模型,且有报道称TPA能有到小鼠皮肤中PPARδ表达的上调,在体外实验中TPA常被用于诱导原代角质细胞的分化,我们预在本实验中考察PPARδ是否也对TPA作用的原代角质起到一定的调控作用。(3)IMQ乳膏溶液作用于角质细胞是最近被研究者开发并使用的一个银屑病细胞模型,能够更进一步的反应银屑病角质细胞的病理情况,我们预考察PPARδ在该体外细胞模型中所起到的作用。方法:本实验成功提取了人原代角质细胞,并分别成功构建了三种角质细胞炎症模型。利用Q-PCR、WB等方法考察了PPARδ选择性激动剂GW0742和专一性拮抗剂GSK3787在这些模型中对炎症的影响,并进一步考察了PPARδ在这一过程中的作用机制。结果:(1)通过实验在众多的PPARδ响应基因中确定了改变最大的一个基因:脂质分化相关蛋白(Adipose differentiation-related protein,ADRP),并以它作为后续实验中考察PPARδ是否激活的一个指标;(2)PPARδ激动剂GW0742能加剧IL-17A、IL-22或TNF-α诱导的原代角质细胞炎症情况;(3)TPA诱导的原代角质细胞炎症情况不依赖于PPARδ通路,但我们发现TPA+GW0742能共同激活人黑素瘤缺乏因子2(Absent in Melanoma 2,AIM-2)的表达,而最近有报道称AIM-2在银屑病的发展中占有一定的作用。(4)PPARδ抑制剂能缓解IMQ诱导的原代角质细胞炎症情况;结论:本部分实验在细胞模型上验证了PPARδ在银屑病相关炎症方面的作用,首先PPARδ的拮抗剂能降低IL-17、IL-22或TNF-α诱导的炎症因子的分泌。TPA诱导的原代角质细胞炎症虽然不依赖于PPARδ,但是当TPA和PPARδ的激动剂同时存在时,AIM-2的表达被上调,这说明PPARδ还能在一定的程度上激活一些通路来协同其它的刺激因素产生其原来所没有的作用,PPARδ的这一现象也与之前报道的其能调控丰富的下游基因的功能相一致。此外,IMQ诱导的原代角质细胞炎症能够被PPARδ的激动剂加强,同时能被PPARδ的抑制剂减弱。第三部分PPARδ在体内银屑病样小鼠模型中的作用及其机制研究目的:前面实验表明PPARδ对于IMQ诱导的原代细胞炎症起到了一定的作用,且PPARδ在银屑病皮损处高表达,为了考察PPARδ在体内银屑病小鼠模型中的作用及其作用机制,设计这一部分实验。方法:首先配制PPARδ的体内注射溶液,在IMQ刺激前2 h、刺激后4 h、8 h注射入小鼠皮下,空白组用PBS代替注射。连续6天后,处死小鼠取材,皮肤均分为4份,一份检测Q-PCR,一份检测WB,一份检测IHC,一份用作流式细胞术检测使用。通过HE染色、IHC染色、Q-PCR、流式细胞术(Flow Cytometry,FCM)等技术,我们观察了小鼠皮肤的病理情况并检测了小鼠皮肤中银屑病相关炎症因子及炎症细胞的变化情况。对于PPARδ作用机制的研究,我们将不同组的小鼠皮肤提取蛋白,进行WB实验,筛选信号通路。结果:PPARδ专一性拮抗剂GSK3787能下调IMQ引起的表皮棘层肥厚现象,同时从HE的结果上看,真皮层的浸润细胞也有所降低。GSK3787还能降低IMQ引起的IL-17a、IL-23、IL-22和IL-1b等的表达升高,并且GSK3787的这些作用呈浓度和时间依赖性。此外,TCRβ+IL-17A+及TCRγδ+IL-17A+(T17+)细胞在IMQ+GSK组也较IMQ组有所下调。对于PPARδ在IMQ模型中作用机制的研究,我们发现PI3K-AKT及STAT3信号通路在模型组小鼠皮损中表达上调,我们进一步注射该通路蛋白的抑制剂,发现PPARδ的作用被减弱。结论:PPARδ拮抗剂能够缓解IMQ引起的银屑病样皮损症状,且能降低银屑病相关炎症因子及炎症细胞的表达,提示其可能作为一种潜在的银屑病治疗新靶点。IMQ诱导的银屑病样小鼠皮肤炎症中,PPARδ是通过PI3K-AKT及STAT3通路来发挥作用的。第四部分阻断Alox8能减轻IMQ在小鼠皮肤上引起的银屑病样反应目的:之前的实验中已经较为深入地探明了PPARδ在银屑病发病中的作用,但是PPARδ的内源性配体还不是很明确。虽然我们在第一部分中阐明,Alox8/8s-HETE是PPARδ潜在的上游配体中表达最高的一对,且有相关研究表明,Alox8/8s-HETE可能在皮肤炎症性疾病的发生中起到一定的作用。但是它们是否真的在体内对PPARδ有这调控的作用我们还不能肯定,为了进一步考察PPARδ的上游配体Alox8对于PPARδ的潜在调控作用,进行了以下实验。方法:目前由于没有市售的PPARδ中和性抗体,为了方便Alox8体内的阻断,我们构建了慢病毒包装的Alox8 sh RNA干扰序列。我们首先考察了用Alox8 sh RNA干扰后,利用Q-PCR实验方法测量了PPARδ各响应基因的激活情况并用免疫共沉淀的方法考察了PPARδ与RXRa结合情况的变化。随后,我们又进一步利用Q-PCR法、WB法和FCM的方法考察了Alox8 sh RNA干扰后,小鼠皮肤中总体炎症情况的改变以及银屑病相关炎症蛋白表达的变化以及炎症细胞数量的改变。结果:体内Alox8 sh RNA干扰后,Alox8蛋白的表达量较阴性对照序列有所下降,此外,小鼠皮肤内PPARδ各响应基因的表达均下调,其中以ADRP的表达下降的最多,与体外实验的趋势相近。Alox8的干扰还使得小鼠皮肤中的总体炎症情况有所缓解。最后Alox8 sh RNA-IMQ小鼠皮肤中的银屑病相关炎症蛋白的表达和炎症细胞的浸润都较IMQ模型组中有所减少。结论:以上实验结果表明,Alox8是PPARδ在银屑病发病中发挥重要作用的关键性配体。对Alox8的干扰不仅能阻断PPARδ的作用,且没有发现明显的不良反应,表明Alox8对于PPARδ的作用可能比较单一,是银屑病治疗的另一个潜在的治疗靶点。
[Abstract]:Psoriasis is a spontaneous autoimmune disease that affects about 2% of the population around the world and has a low incidence in China, but China has a large population base, with about 500-800 million patients with psoriasis. As psoriasis is not yet radical, and it is especially easy to relapse, it has caused serious damage to the patients and their families. Im Iquimod, IMQ) was originally used to treat human skin cancer and external genital / perianal warts and other diseases, which were developed and used to induce psoriasis like symptoms in the skin of mice. The model can induce most of the symptoms of psoriasis, and the model is easy to be prepared and quickly become an important model for the study of psoriasis. The peroxisome proliferator activated receptor (peroxisome proliferator-activated receptors, PPARs) belongs to the II type nuclear receptor superfamily, and can be divided into 3 subtypes (PPAR alpha / gamma / delta).PPAR Delta in the metabolic syndrome, atherosclerosis, lipid metabolism disorder and other diseases. Previous experiments found PPAR Delta in human psoriasis. The high expression of skin lesions and IMQ induced psoriasis in mice suggests that it may play a role in the development of psoriasis. At present, the treatment of psoriasis is not very good and can not be cured thoroughly. This experiment was conducted with the aid of IMQ induced psoriasis like mice and human keratinocytes for a series of experiments for PPAR Delta. The role of psoriasis provides more powerful and direct evidence to provide new therapeutic targets for the treatment of psoriasis. Part 1 the expression of PPAR Delta in psoriatic lesions: To investigate the expression of PPAR Delta in the skin and skin of psoriasis in humans and mice and to provide the role of PPAR Delta in psoriasis. Methods: the skin, normal skin and IMQ stimulated skin of healthy skin and silver chip patients, immunohistochemistry (IHC), real-time fluorescent quantitative PCR (quantitative polymerase chain reaction, Q-PCR) and protein immunoblotting (Western Blot, WB) experiment were carried out to investigate the normal skin and the normal skin. The expression difference in epidermis and dermis in psoriatic lesions and the specific expression of PPAR Delta in the skin lesions of psoriasis. Results: the expression of PPAR Delta in the skin lesions of human psoriasis and IMQ stimulated skin in mice was higher than that in healthy skin and normal mice. The main table of PPAR Delta in human skin lesions was observed by IHC staining. The expression of PPAR Delta in human and mouse skin is mainly expressed in the epidermis, while the content of.PPAR Delta in the dermis is rarely expressed in the skin of the healthy human skin, and it is also rarely expressed in the skin lesions of several other skin diseases, indicating the relative specificity of PPAR delta expressed in the skin lesions of psoriasis. Conclusion: PPAR Delta in psoriasis and IMQ induced psoriasis in the skin lesions of mice, indicating that PPAR delta may play an important role in the pathogenesis of psoriasis. Second part of the function of PPAR Delta for the secretion of cytokine in the primary keratinocytes in vitro: investigation of PPAR Delta in different cellular inflammatory models In this part, we have used three types of cell models that are more closely related to psoriasis. (1) cytokines such as interleukin (IL) -17, IL-22 and tumor necrosis factor alpha (tumor necrosis factor, TNF- a) play an important role in the development of psoriasis, we have explored the PP. The effect of AR Delta on the inflammatory response of keratinocytes under the action of these cytokines. (2) 12-O- fourteen alkyl acetate -13 (12-O-Tetradecanoylphorbol-13-acetate, TPA) is also used to induce psoriasis like model in mice in vivo, and it is reported that TPA can increase the expression of PPAR Delta in mouse skin, and TPA often in vitro. It is used to induce differentiation of primary keratinocytes. In this experiment, we investigate whether PPAR delta may also play a role in regulating the primary horniness of TPA. (3) the IMQ cream solution acts on keratinocytes as a psoriatic cell model developed and used recently by researchers to further respond to psoriatic keratinocytes. We previewed the role of PPAR Delta in this extracorporeal cell model. Methods: the human primary keratinocytes were successfully extracted and three keratinocytes were successfully constructed. The PPAR delta selective agonist GW0742 and the exclusive antagonist GSK3787 were investigated by Q-PCR, WB and other methods. The effect of PPAR Delta on this process was further investigated. Results: (1) the largest one was identified by the experiment in many PPAR delta response genes: the Adipose differentiation-related protein (ADRP), and it was used as a follow-up to investigate whether the PPAR delta was activated. One index: (2) PPAR delta agonist GW0742 can aggravate primary keratinocyte inflammation induced by IL-17A, IL-22 or TNF- alpha; (3) TPA induced primary keratinocyte inflammation does not depend on the PPAR delta pathway, but we found that TPA+GW0742 can co activate the expression of human melanoma deficiency factor 2 (Absent in Melanoma 2, AIM-2), and recently reported It is known that AIM-2 plays a role in the development of psoriasis. (4) PPAR delta inhibitors can relieve IMQ induced primary keratinocyte inflammation. Conclusion: this part of this experiment demonstrated the role of PPAR Delta in psoriasis related inflammation. First, PPAR delta antagonists can reduce the inflammatory factors induced by IL-17, IL-22 or TNF- alpha. The secretion of.TPA induced primary keratinocytes is not dependent on PPAR Delta, but when the activator of TPA and PPAR delta exists simultaneously, the expression of AIM-2 is up-regulated, indicating that PPAR delta can also activate some pathways to produce its original role in conjunction with other stimuli. This phenomenon of PPAR delta is also the same as before. It is reported that it can regulate the functions of the rich downstream genes. In addition, IMQ induced primary keratinocyte inflammation can be enhanced by PPAR delta agonists and can be weakened by the inhibitor of PPAR Delta. The role and mechanism of the third part of PPAR Delta in the mice model of psoriasis like mice and its mechanism: the previous experiment showed that PPAR delta was lured to IMQ. The primary cell inflammation plays a certain role, and PPAR delta is highly expressed in the skin lesions of psoriasis. In order to investigate the role and mechanism of PPAR Delta in the mice model of psoriasis in vivo, this part of the experiment was designed. Method: first, the injection solution of PPAR delta was prepared in vivo, 2 h before IMQ stimulation, 4 h after stimulation, and 8 h injection into the subcutaneous of mice. The blank group replaced the injection with PBS for 6 days. The mice were sacrificed for 6 days. The skin was divided into 4 parts, one was Q-PCR, one was tested for WB, one was IHC, and one was used for flow cytometry. We observed the pathology of the skin of mice by HE staining, IHC staining, Q-PCR, flow cytometry (Flow Cytometry, FCM) and other techniques. The changes of inflammatory factors and inflammatory cells related to psoriasis in the skin of mice. For the study of the mechanism of PPAR delta action, we extracted protein from different groups of mice, carried out WB experiments and screened signal pathways. Results: the PPAR delta specific antagonist GSK3787 could downregulate the hypertrophy of the epidermal spinous layer caused by IMQ and the result of HE. In the dermis, the infiltrating cells of the dermis also reduced.GSK3787 and decreased the expression of IL-17a, IL-23, IL-22 and IL-1b caused by IMQ, and the effects of GSK3787 were concentration and time dependent. Furthermore, TCR beta +IL-17A+ and TCR gamma +IL-17A+ (T17+) cells were also down down in the group. We found that the PI3K-AKT and STAT3 signaling pathways were up-regulated in the skin lesions of the model mice. We further injected the inhibitor of this pathway protein and found that the effect of PPAR delta was weakened. Conclusion: PPAR delta antagonists can relieve the symptoms of psoriasis like skin lesions caused by IMQ and reduce the inflammatory factors associated with psoriasis and the fine inflammation. Cell expression, suggesting that PPAR delta may play a role in the skin inflammation of psoriasis like mice induced by.IMQ, a new target for psoriasis treatment. The fourth blocking of Alox8 can alleviate the psoriasis like response to IMQ in the skin of mice: the previous experiments have been more thorough. The role of PPAR Delta in the pathogenesis of psoriasis is explored, but the endogenous ligands of PPAR delta are not very clear. Although we have stated in the first part that Alox8/8s-HETE is the highest expression of the PPAR delta potential upstream ligand, the relevant studies suggest that Alox8/8s-HETE may play a certain role in the occurrence of inflammatory diseases of the skin. But we are not sure whether they really play a role in the regulation of PPAR Delta in the body. In order to further investigate the potential regulation of the upstream ligand of PPAR Delta, Alox8 for PPAR Delta, the following experiments are carried out. Method: we are currently constructing the PPAR delta neutralization anti body, in order to facilitate the blocking of Alox8 in the body. The Alox8 sh RNA interference sequence of the lentivirus package. We first examined the Alox8 sh RNA interference and measured the activation of each response gene of PPAR Delta using the Q-PCR experimental method and examined the changes in the combination of PPAR Delta and RXRa with the method of immunoprecipitation. After the interference of Alox8 sh RNA, the overall inflammation in the skin of mice and the changes in the expression of inflammatory proteins related to psoriasis and the changes in the number of inflammatory cells. Results: after the interference of Alox8 sh RNA in the body, the expression of Alox8 protein decreased more than that of the negative control sequence. In addition, the expression of the PPAR delta response genes in the skin of the mice was all lower. The expression of ADRP decreased most, and the.Alox8 interference with the trend of in vitro experiments also made the overall inflammation in the skin of the mice relieved. Finally, the expression of inflammatory proteins related to psoriasis and the infiltration of inflammatory cells in the skin of Alox8 sh RNA-IMQ mice were less than those in the IMQ model group. The results show that Alox8 is the key ligand for PPAR delta to play an important role in the pathogenesis of psoriasis. The interference with Alox8 not only blocks the effect of PPAR Delta, but also does not find obvious adverse reactions. It indicates that the effect of Alox8 on PPAR delta may be relatively single, and it is another potential therapeutic target for the treatment of psoriasis.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R758.63

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