IL-31与NT-4在特应性皮炎发病中相互关系及其致痒机制研究

发布时间：2018-03-30 16:26

本文选题：白介素-31　切入点：神经营养素-4　出处：《重庆医科大学》2012年硕士论文

【摘要】：背景特应性皮炎(atopic dermatitis, AD)是一种与遗传过敏素质有关的慢性复发性、瘙痒性、炎症性皮肤疾病。瘙痒是AD主要特征之一,“瘙痒-搔抓”恶性循环在AD皮肤炎症的加重和持续中占有重要地位,控制瘙痒是AD治疗的主要目标。关于AD瘙痒的病理生理涉及皮肤感觉神经纤维、神经肽、神经营养素、炎症细胞和炎症因子等多个方面,但至今AD瘙痒发生的确切机制并不清楚。白介素-31(IL-31)是近年发现的一种新型白介素,主要由活化的Th2细胞产生,通过与IL-31受体A(IL-31RA)和抑瘤素M受体β(OSMR B)组成的功能受体复合物结合而发挥生物学作用。前期研究发现IL-31不仅参与AD发病而且与AD的瘙痒发生有关,但IL-31在AD瘙痒的作用机制尚不清楚。目的本研究通过检测不同严重程度AD患儿血清IL-31及NT-4水平,探讨IL-31及神经营养素在AD发病中的作用及其与AD严重程度的相关性。在此基础上,采用卵清蛋白(OVA)经皮肤反复致敏诱导的AD小鼠模型,通过检测AD小鼠皮损处IL-31mRNA、IL-31RA、OSMRβ及神经营养素NT-4受体TrkB的表达及分布,探讨IL-31及NT-4参与AD瘙痒的可能机制。方法 1.符合Hanifin-Rajka诊断标准的AD患儿69例,采用SCORAD评分评估其疾病严重程度,ELISA方法测定患儿血清IL-31及NT-4水平。 2.使用OVA经皮肤反复致敏诱导建立AD小鼠模型,采用实时定量RT-PCR法检测AD小鼠皮损处IL-31mRNA表达,同时ELISA法测定小鼠血清IL-31和NT-4水平。免疫组化法和蛋白印迹法(Western blot)检测皮损处IL-31受体IL-31RA、OSMRβ及神经营养素NT-4受体TrkB的分布及表达情况。结果 1.本组69例AD患儿疾病严重程度评分SCORAD53.56±14.44,其中轻中度(SCORAD"f50)27例(39.1%),SCORAD39.78±8.92,重度(SCORAD50)42例(60.9%),SCORAD62.42±9.48。 2.AD患儿血清IL-3134.73±13.33pg/ml,显著高于对照组(20.89±8.90pg/ml; p0.001),其中轻中度组28.52±13.00pg/ml,显著高于对照组(p0.05),重度组38.72±12.08pg/ml,显著高于对照组(p0.001)及轻中度组(p0.01)。 3.AD患儿血清NT-4中位数8.84ng/ml (1.55-45.75),显著高于对照组(2.95ng/ml(0.82-21.12)；p0.01)；轻中度组10.61±9.17ng/ml及重度组14.73±11.90ng/ml,均显著高于对照组(p0.05；p0.01)。 3.AD患儿SCORAD评分与血清IL-31水平呈正相关(r=0.557,p0.001)。AD患儿血清IL-31水平与血清NT-4水平呈正相关(r=0.515,p0.001)。在重度AD患儿中,血清NT-4水平与SCORAD呈正相关(r=0.327,p0.05)。 4.AD小鼠模型在致敏第6周,接触部位明显水肿,肥厚,皮损组织HE染色镜下见AD小鼠表皮颗粒层增生,棘层肥厚,真皮层血管扩张充血,胶原纤维显著增生,并可见淋巴细胞、中性粒细胞及嗜酸性粒细胞等炎症细胞浸润,对照组基本正常。说明AD小鼠模型构建成功。 5.AD小鼠皮损组织IL-31mRNA表达显著增强,是对照组的13.89倍(p0.01)。 6.AD小鼠血清IL-31水平(171.63±14.15pg/ml)显著高于对照组152.15±14.19pg/ml,p0.05),AD小鼠血清NT-4水平(115.47±16.50pg/ml)较对照组(112.99±11.20pg/ml)差异无统计学意义(p0.05)。 7.AD小鼠皮损处可见IL-31RA在角质形成细胞,毛囊细胞及巨噬细胞胞浆内均呈强阳性表达,OSMRβ主要表达于角质形成细胞和毛囊细胞胞浆内,同时神经营养素NT-4受体TrkB在角质形成细胞胞浆及神经纤维内呈阳性表达。与对照组比较,AD小鼠皮损处IL-31RA蛋白表达显著增强(0.78±0.43vs0.40±0.19；p0.05),TrkB蛋白表达增强(0.05±0.24vs0.03±0.16；p0.05),而OSMRP蛋白表达量无统计学差异(0.40±0.21vs0.28±0.17,p0.05)。结论 1.AD患儿外周血IL-31水平明显升高且与疾病严重程度呈正相关。AD小鼠血清IL-31水平与皮损处IL-31mRNA表达均显著升高,证明IL-31与AD发生密切相关,是反映AD疾病严重程度的重要指标。 2.AD患儿外周血NT-4水平升高,同时血清IL-31与NT-4水平也呈正相关关系,说明神经营养素NT-4可能在AD的发病中起重要作用。 3.在AD小鼠模型皮损处IL-31受体IL-31RA和OSMRP表达在角质形成细胞胞浆内,神经营养素NT-4受体TrkB在神经纤维内呈阳性表达,同时IL-31RA和TrkB蛋白表达显著增强。由此推测,IL-31可能经由位于角质形成细胞内的IL-31RA/OSMRβ功能受体复合物,影响角质形成细胞功能,促进角质形成细胞分泌NT-4,通过与神经纤维内的受体TrkB结合,参与AD瘙痒的病理生理过程。
[Abstract]:background
Atopic dermatitis (atopic dermatitis AD) is a kind of related genetic anaphylactic predisposition to recurrent chronic itching, itching, inflammatory skin disease. AD is one of the main features of the "itch scratch" vicious spiral AD plays an important role in the inflammation of the skin and increase continuously, to control the itching is the main goal of AD about AD treatment. Pathological itching relates to cutaneous sensory nerve fibers, neuropeptides, neurotrophins, multiple inflammatory cells and inflammatory factors, but the exact mechanism is still AD itching occurs is not clear.
Interleukin -31 (IL-31) is a new type of interleukin found in recent years, mainly produced by activated Th2 cells through IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR B) functional receptor complex combination play a biological role. Previous study found that IL-31 is not only involved in the pathogenesis of AD. And AD itching occurs, but IL-31 in the mechanism of AD itching is not clear.
objective
This study through the detection of different severity of AD serum IL-31 and NT-4 levels, to explore the role of IL-31 and neurotrophins in the pathogenesis of AD and its correlation with the severity of AD. On this basis, using ovalbumin (OVA) by AD mice model induced by repeated skin sensitization, mice skin lesions detected by AD IL-31mRNA, IL-31RA the expression and distribution of OSMR, NT-4 and beta neurotrophin receptor TrkB, and explore the possible mechanism of IL-31 and NT-4 in AD pruritus.
Method
1. of the 69 children with Hanifin-Rajka diagnostic criteria, the severity of the disease was evaluated by SCORAD score, and the level of serum IL-31 and NT-4 was measured by ELISA method.
2. using OVA skin sensitization induced by repeated AD mice model was established, using quantitative real-time RT-PCR assay for detection of AD lesions in mice IL-31mRNA expression, serum IL-31 and NT-4 levels in mice and the ELISA method. Immunohistochemical method and Western blotting (Western blot) IL-31 IL-31RA lesions by detecting the expression and distribution of OSMR and beta. NT-4 neurotrophin receptor TrkB.
Result
1., in the 69 cases of AD, the severity score of SCORAD53.56 was 14.44, including mild to moderate (SCORAD, F50), 27 cases (39.1%), SCORAD39.78 + 8.92, severe (SCORAD50) 42 cases (60.9%), SCORAD62.42 + 9.48..
The serum IL-3134.73 + 13.33pg/ml in children with 2.AD was significantly higher than that in the control group (20.89 + 8.90pg/ml, p0.001), in which mild and moderate group was 28.52 + 13.00pg/ml, which was significantly higher than that of the control group (P0.05), and the severe group was 38.72 + 12.08pg/ml, which was significantly higher than that of the control group (p0.001) and mild to moderate group (P0.01).
The median serum NT-4 level of 8.84ng/ml in children with 3.AD (1.55-45.75) was significantly higher than that in the control group (2.95ng/ml (0.82-21.12); P0.01); in mild to moderate group, 10.61 + 9.17ng/ml and severe group were 14.73 + 11.90ng/ml, which were significantly higher than those in the control group (P0.05, P0.01).
There was a positive correlation between SCORAD score and serum IL-31 level in children with 3.AD (r=0.557, p0.001). There was a positive correlation between serum IL-31 level and serum NT-4 level in children with.AD (r=0.515, p0.001).
In a mouse model of 4.AD sensitized for sixth weeks, contact area of edema, hypertrophy, skin lesions and HE staining AD mouse epidermal granular layer hyperplasia, acanthosis, dermal vascular hyperemia, collagen fiber hyperplasia, inflammatory cells and lymphocytes, neutrophils and eosinophils infiltration in the control group normal. AD mouse model was successfully constructed.
The expression of IL-31mRNA in the skin tissue of 5.AD mice was significantly enhanced, 13.89 times as much as that of the control group (P0.01).
The serum IL-31 level of 6.AD mice (171.63 + 14.15pg/ml) was significantly higher than that of the control group (152.15 + 14.19pg/ml, P0.05). The serum NT-4 level of AD mice (115.47 + 16.50pg/ml) was not significantly different from that of the control group (112.99 + 11.20pg/ml) (P0.05).
7.AD mouse skin lesions visible IL-31RA in keratinocytes, hair follicle cells and macrophages in the cytoplasm showed strong positive expression of OSMR beta is mainly expressed in keratinocytes and hair follicle cells in the cytoplasm, and NT-4 neurotrophin receptor TrkB forming cell cytoplasm and in the nerve fibers positive expression in keratinocytes compared with the control group. The expression of AD in lesions of mice, IL-31RA protein significantly increased (0.78 + 0.43vs0.40 + 0.19; P0.05), enhanced expression of TrkB (0.05 + 0.24vs0.03 + 0.16; P0.05), but no significant difference between the expression of OSMRP (0.40 + 0.21vs0.28 + 0.17, P0.05).
conclusion
The level of IL-31 in peripheral blood of children with 1.AD increased significantly, and positively correlated with the severity of disease. The level of IL-31 in serum and the expression of IL-31mRNA in skin lesions of.AD mice increased significantly, indicating that IL-31 is closely related to AD and is an important index reflecting the severity of AD disease.
The level of NT-4 in peripheral blood of children with 2.AD increased. Meanwhile, the level of serum IL-31 was positively correlated with NT-4 level, indicating that neurotrophin NT-4 might play an important role in the pathogenesis of AD.
3. in a mouse model of AD lesions of IL-31 receptor IL-31RA and OSMRP expression in keratinocytes cytoplasm, neurotrophin receptor NT-4 positive expression of TrkB in nerve fibers, co expression of IL-31RA and TrkB protein increased significantly. Therefore, IL-31 may be located in keratinocytes via IL-31RA/OSMR beta receptor complex functions in cells, cells effect of HaCaT keratinocytes, promote the secretion of NT-4, with the nerve fibers within the receptor TrkB, involved in the pathophysiological process of AD pruritus.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R758.2

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