【作者】 郭红卫；

【导师】 郝飞；

【作者基本信息】 第三军医大学 ， 皮肤性病学， 2010， 博士

【摘要】 斑秃(alopecia areata, AA)是在一定遗传背景下,多种因素诱发的局限于毛囊的自身免疫性疾病。应激可诱发和加重斑秃,目前认为斑秃是一种应激相关的炎症性皮肤病。应激反应与下丘脑-垂体-肾上腺轴(hypothalamic-pituitary-adrenal axis, HPA axis)、交感神经系统、免疫系统紧密相关,其中被称为应激轴(stress axis)的HPA轴,是介导应激反应的重要途径。HPA轴系统反应缺陷将导致糖皮质激素缺乏状态,可增加自身免疫性疾病的易感性,是应激相关的自身免疫性疾病的重要致病因素之一。目前发现除了经典的中枢HPA轴,外周组织皮肤及其附属器也存在着与中枢HPA轴相似的HPA轴。皮肤及毛囊的HPA轴与中枢HPA轴不但具有相同的环路分子而且具有相似的神经内分泌功能,与神经递质、其它神经肽以及神经营养素共同调控生理病理状态下皮肤的神经内分泌及免疫功能。近来一些研究显示,中枢及外周的HPA轴活动缺陷均参与应激相关的斑秃的发病,但其具体机制尚未完全阐明。HPA轴的组成分子也被称为应激激素(stress hormones),包括促皮质激素释放激素(corticotropin-releasing hormone,CRH)、促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)和皮质醇(cortisol)。CRH信号必须经过ACTH/MC2R途径刺激皮质醇生成,ACTH被称为“内分泌第二信使”;而且ACTH在几个黑素皮质激素原肽(proopiomelanocortin, POMC)衍生肽中,是主要调节毛发生长的POMC衍生肽,因此ACTH及其特异性的受体黑素皮质素受体2(melanocortin receptor type 2,MC2R或adrenocorticotropic hormone receptor, ACTHR)在应激相关的斑秃中可能发挥重要作用。我们对MC2R基因及其蛋白在斑秃皮损中的表达情况进行研究,以阐明HPA轴在斑秃发病中的可能机制,并探讨影响MC2R分子功能的可能调控机制。目前认为斑秃也是一多基因决定的复杂性疾病,因此斑秃的遗传学研究也是阐释斑秃发病机制的途径之一。斑秃家族的全基因扫描发现最大多点LOD值位于18号染色体的10-30CM之间,我们通过GenBank搜索该区域的基因,发现MC2R恰恰位于这个区域。已证实MC2R启动子一些位点的多态性可降低ACTH/MC2R连接敏感性,导致ACTH/MC2R介导的HPA轴信号传导障碍,皮质醇生产减少。认为MC2R基因的这种单个基因的单个硷基突变导致肾上腺皮质醇生成减少,是机体HPA轴活动调控的重要机制之一。中国汉族人群中MC2R启动子这些位点的多态性降低ACTH与MC2R结合能力的研究已有报道。综合斑秃患者异常的HPA轴活动和MC2R基因结构功能特点,我们运用病例对照研究方法,对重庆地区斑秃患者MC2R基因启动子两个位点的多态性进行了检测,统计学分析MC2R基因启动子多态性与斑秃的关联性,尝试阐明影响斑秃HPA轴异常的可能因素。首先我们应用Thomas Holmes和Richard Rahe社会再适应量表调查斑秃患者病情活动期前6个月的应激状态,探讨应激生活事件与斑秃发生风险的相关性。然后,采用免疫组织化学技术检测了MC2R蛋白在斑秃皮损及健康对照头皮组织的原位表达情况,并且应用Western blotting方法对斑秃皮损与健康对照头皮组织MC2R蛋白的总体水平做了定量检测,定位定量地对MC2R蛋白水平在斑秃皮损中的改变进行了全面研究。进而,我们进行了斑秃皮损与健康对照头皮组织MC2R的基因水平的检测与比较研究,求证MC2R是否在头皮组织中合成,探讨斑秃皮损是否存在MC2R基因水平的改变。最后,我们用连接酶检测方法对MC2R启动子区的两个位点的SNP做了研究,从遗传学角度探讨MC2R与斑秃发病的关联性。研究主要结果如下:1.50.95%的斑秃患者在病情活动前6个月经历过3个及以上的应激生活事件,32.05%的健康对照在填表前6个月经历过3个及以上的应激生活事件,斑秃患者罹患应激事件百分率明显高于健康对照(P<0.001);斑秃患者的SRRS评分显著高于健康对照组(Z=6.628, P<0.001),斑秃患者与健康对照人群在应激感受上存在显著差异。经多因素logistic回归分析,睡眠改变、个人习惯改变、家庭问题、学习工作问题及财政问题是与斑秃发生及加重相关的主要应激生活事件,提示应激与斑秃活动相关。2.免疫组化示:MC2R在表皮、附属器及真皮组织中均广泛表达,在附属器表达最明显,主要为细胞膜上与细胞浆内表达。斑秃患者皮损MC2R表达较健康对照头皮在皮肤全层组织,包括表皮、毛囊、皮脂腺及真皮中的表达普遍降低(P<0.05),尤其在毛囊的外毛根鞘(outer root sheath, ORS)、毛母质及表皮的棘细胞层与颗粒细胞层中表达降低显著。MC2R蛋白表达在斑秃表皮及间充质组织均广泛改变,提示MC2R蛋白可能参与斑秃皮损包括表皮及真皮成分的组织病理学改变,显著的表皮表达改变提示MC2R在斑秃表皮病理改变中发挥更重要的作用。3.Western blotting示:斑秃患者皮损中MC2R总蛋白水平较健康头皮组织表达降低(P<0.05),半定量显示了斑秃皮损与健康对照头皮MC2R蛋白表达差异。4.MC2R mRNA在健康头皮的表达量不高,而斑秃皮损MC2R mRNA表达较健康头皮表达增加将近5倍,统计学检验具有显著性(P<0.01)。基于3与4的结果,我们认为斑秃皮损中MC2R基因与蛋白水平均发生改变,MC2R基因与蛋白水平表达的不一致性,提示转录后调控可能在MC2R的基因表达中发挥重要作用,MC2R mRNA不能完全有效翻译为MC2R蛋白可能是形成斑秃组织中HPA轴功能缺陷的关键因素之一。5.利用生物信息学方法,选择MC2R基因启动子的-759A>C(rs1893220)及-2T>C(rs79533878)位点进行SNP研究。首先证实这些位点在中国人群确实存在基因多态性,然后比较发现斑秃患者与正常对照MC2R基因-759A>C、-2T>C等位基因频率无明显统计学差异(P>0.05)。但当我们将斑秃患者按照应激状态分层分析,发现明显应激组的斑秃患者与健康对照者MC2R基因的-2T>C的等位基因频率相比有统计学差异(41.1%比27.8%,P=0.009, OR=1.252, 95%CI= 1.045～1.501)。将结果以非参数Logistic回归校正年龄及性别因素后分析,显示明显应激组的斑秃患者与MC2R基因的-2T>C位点的C等位基因携带有相关性(P=0.002, OR=1.576, 95%CI=1.148～2.162)。我们的研究结果提示MC2R基因-2T>C多态性与斑秃患者的应激感受有关联性,推测MC2R基因-2T>C位点T→C单个硷基的突变降低了ACTH/MC2R连接敏感性,从而不能有效下传HPA系统的CRH信号,造成糖皮质激素生成不足,使自身免疫性疾病斑秃的易感性增加,斑秃患者的精神症状易感性增加。综上所述:我们的研究发现应激生活事件是斑秃发生的风险因素之一,斑秃患者与健康对照人群在应激感受上存在显著性差异。斑秃皮损区MC2R基因及蛋白表达水平与健康对照头皮组织表达有明显差异,HPA轴表达异常参与斑秃的发病。过度应激刺激下,为维护机体正常机能,HPA轴首先发生过度的中枢及外周应激反应,表现为MC2R基因在皮肤中合成增加。而缺陷的转录后调控使MC2R基因不能完全表达为MC2R蛋白,导致MC2R蛋白表达降低。下调的MC2R蛋白致使HPA轴信号不能有效下传,糖皮质激素生成缺乏,增加自身免疫性疾病斑秃的易感性。我们推想MC2R的转录后调控机制可能是形成斑秃组织中缺陷的HPA轴功能的关键机制之一。MC2R基因启动子的某些位点能够影响ACTH/MC2R连接敏感性导致缺陷的HPA轴活动。我们调查发现MC2R基因启动子-2T>C(rs79533878)位点SNP与斑秃患者的应激感受关联,推测MC2R基因启动子区-2位点基因突变能够引起ACTH/MC2R连接敏感性降低,导致HPA轴CRH信号不能有效下传,致使糖皮质激素生成缺陷,增加自身免疫病及炎症的易感性,增加个体精神症状易感性,从遗传学角度探讨了MC2R与应激相关的斑秃患者缺陷的HPA轴的关联性。总之,我们的研究对应激相关的斑秃发病机制的阐明以及从神经内分泌免疫遗传学方面制定防治斑秃措施提供了理论依据。更多还原

【Abstract】 Alopeica areata (AA) is a hair follicle-specific autoimmune disease with genetic predisposition and environmental trigger and AA is gradually accepted as an inflammatory skin disease responding to stress. Stress response is mediated by the stress system involved hypothalamic-pituitary-adrenal axis (HPA axis), sympathetic nervous system and immune system, in which HPA axis called stress axis plays a cardinal role. Growing studies showed that a decreased HPA axis response could cause increased susceptibility to autoimmune and inflammatory disease just as the glucocorticoid-deficient state.Except the classical central HPA axis, CRF and related peptides can act as local modulators of stress in the peripheral organs such as skin, gestational tissues, immune system, pancreas, liver, gastrointestinal tract, skeletal muscle, heart, lung and endocrine organs (ovaries, testes, adrenals, thyroid glands and ocular tissue. It has been grdually acknowledged that skin and hair follicle display peripheral HPA-axis-like signaling systems and the equivalent components of the HPA axis coordinate with neurotransmitters, neuropeptides and neurotrophins effectively modulate skin and immune cell functions such as cell proliferation, cytokine production and the hair cycle under normal and pathological conditions. Recently some studies showed that an insufficient HPA axis response involved in the pathogenesis of AA in spite that the pathology of stress-associated AA has not been well recognized.The HPA axis is organized in a hierarchical manner with feedback operating at several points along the axis. The elements of HPA axis titled stress hormones include corticotropin-releasing hormone (CRH), adrenocorticotropin (ACTH) and corticosteroids. Stress stimulates the HPA axis activity with an elevated secretion of CRH leading to increased ACTH secretion and corticosteroid production. These stress hormones binding with their cognate receptors activate the signaling hierarchy to respond stress. ACTH intermediates the HPA axis from CRH signal to cortisol secretion and is recognized to have a strongest effect on hair growth among all of the POMC peptides, so the ACTH specific recptor melanocortin receptor type 2 (MC2R or adrenocorticotropic hormone receptor, ACTHR) with its binding affinity restricted to ACTH may present a link between the physiological response to stressors and hair follicle-restricted immunological inflammation AA. To answer this critical question, we assessed AA lesions and normal human scalp tissue for the expression of MC2R.It is now generally accepted that AA fits the paradigm of a complex genetic trait, therefore genetic studies can be applied in study on AA pathogenesis. A genome-wide search revealed that a maximum multipoint LOD (logarithm of odds) score with most significant susceptibility position is in the region from 10cM to 30cM on chromosome 18. We searched genes of this region in GenBank and found the MC2R is just located in the range. Emerging evidences that single base mutation in the promoter region of MC2R gene can result in significantly reduced adrenal ACTH sensitivity and lowed glucocorticoid serum levels during ACTH infusion indicates that the polymorphism of the positions of the ACTH receptor gene may have a determining role in controlling glucocorticoid production in human. Furthermore several polymorphisms in promoter region of MC2R gene resulting in a lower ligand affinity with ACTH has also been reported in Chinese Han people. So our research is to investigate whether there was any association between MC2R promoter SNPs and stress-associated AA to decode the genetic mechanism of AA.Firstly we used Thomas Holmes and Richard Rahe’s social readjustment rating scale to assess the association of psychosocial stress with AA patients, secondly MC2R protein was determined by immunohistochemistry in situ and western blotting quantificationally, thirdly MC2R mRNA level was detected with quantitative real-time RT-PCR, finally two SNPs were assessed by LDR at positions -759A>C and -2T>C in the MC2R promoter.The main results and conclusions were summarized as follows:1. 50.95% of patients with AA experienced at least three stressful events at the time and up to 6 months prior hair loss compared with 32.05% of controls within the six months before the enrolling (P<0.001). The SRRS results show patients with AA have a higher depression scores than controls (Z=6.628,P<0.01). The main stressful life events at risks for AA patients were changes of sleep, modification of habits, family troubles, problems of work and school as well as involvement of finance by logistic regression analysis. Our studies demonstrated that a significant elevation of psychological stress was felt by AA patients and there was a closely relationshiop between risk of AA and severe perception of some main stressful events in Chinese population.2. Immunohistochemistry demonstrated the MC2R protein was widely expressed on cell membrane and in cytoplasm in nearly all cutaneous compartments and was most prominently detectable in adnexal structures. Average MC2R protein levels in every section of skin were lower in AA lesions than in scalp tissue from normal controls and the most differences were showed in outer root sheath, hair bulb and the stratum spinosum and stratum granulosum of epidermis. The reduced expression of MC2R in the epithelium of AA lesions is remarkable. Thus we suggested that the modified expression of MC2R is almost comprehensively participated in the histopathology of AA and particularly involved in epithelial pathology of AA.3. Western blotting results demonstrated that the expression of total MC2R protein was decreased in lesions of AA than scalp tissues from healthy controls (P<0.05). The data confirmed the findings of immunohistochemistry quantificationally.4. There was low expression of MC2R mRNA in normal scalp tissue. About 5-fold increased MC2R mRNA expression in lesion from patients with AA was detected compared with that in scalp tissue from cortols (P<0.01).The findings 3, 4 demonstrated that there were robust differences in MC2R expression between healthy controls with AA patients. While mRNA levels were increased in lesions from AA patients compared with scalp tissues from normal controls, the protein levers of MC2R were decreased. We hypothesized that the contrary expression of MC2R on gene and protein lever just coincided with the evidence that an over-responsive HPA activity coexists with a deficient HPA response in AA. We proposed that the failed translation of MC2R mRNA to protein may be a crucial factor for the local insufficient HPA response in AA.5. Two SNPs were selected at position -759A>C and -2T>C in the MC2R promoter with accession numbers of rs1893220 and rs79533878. There was a significant difference between AA patients with stress and controls in the frequency of the MC2R rs79533878 (-2T>C) genotype CC+TC (41.1% vesus 27.8%,P=0.009, OR=1.252, 95%CI= 1.045～ 1.501). The novel MC2R promoter variants rs79533878 (-2T>C) significantly associated with the stress-associated AA (P=0.002, OR=1.576, 95%CI=1.148～2.162). However the allele frequencies at position -759A>C in the MC2R promoter did not show statistically differences between the patients and control groups. Our findings implicated that the polymorphism of the rs79533878 (-2T>C) in MC2R gene may be one important factor that are correlated with the modified HPA axis response to stress in AA patients in China.In conclusion: Our results demonstrated that AA in some patients was associated with stress. We suggested an over-responsiveness of HPA axis to stress might trigger the first episode of AA considering the reportedly elevated CRH, ACTH and the high MC2R mRNA expressin in lesion of AA, but the lower levels of MC2R protein, resulting in a deficit of ACTH/MC2R activity in skin could attenuate CRH singnal transmission and reduce HPA axis response locally in AA. We proposed the deficient translation of MC2R mRNA to protein may be a key factor for the local insufficient HPA response and shortage of cortisol. Our investigation also showed the novel MC2R promoter variant rs79533878 (-2T>C) significantly associates with some stress-associated AA and we suggested that the polymorphism of the MC2R rs79533878 (-2T>C) in promoter might be one critical factor that influences the altered HPA axis activity in AA. Our research would bring new knowledge into understanding of stress-associated hair-loss and would help design new approaches for the treatment of stress-associated AA in future.更多还原