本文選題：白介素-31　切入點(diǎn)：神經(jīng)營養(yǎng)素-4　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景 特應(yīng)性皮炎(atopic dermatitis, AD)是一種與遺傳過敏素質(zhì)有關(guān)的慢性復(fù)發(fā)性、瘙癢性、炎癥性皮膚疾病。瘙癢是AD主要特征之一,“瘙癢-搔抓”惡性循環(huán)在AD皮膚炎癥的加重和持續(xù)中占有重要地位,控制瘙癢是AD治療的主要目標(biāo)。關(guān)于AD瘙癢的病理生理涉及皮膚感覺神經(jīng)纖維、神經(jīng)肽、神經(jīng)營養(yǎng)素、炎癥細(xì)胞和炎癥因子等多個(gè)方面,但至今AD瘙癢發(fā)生的確切機(jī)制并不清楚。 白介素-31(IL-31)是近年發(fā)現(xiàn)的一種新型白介素,主要由活化的Th2細(xì)胞產(chǎn)生,通過與IL-31受體A(IL-31RA)和抑瘤素M受體β(OSMR B)組成的功能受體復(fù)合物結(jié)合而發(fā)揮生物學(xué)作用。前期研究發(fā)現(xiàn)IL-31不僅參與AD發(fā)病而且與AD的瘙癢發(fā)生有關(guān),但I(xiàn)L-31在AD瘙癢的作用機(jī)制尚不清楚。 目的 本研究通過檢測不同嚴(yán)重程度AD患兒血清IL-31及NT-4水平,探討IL-31及神經(jīng)營養(yǎng)素在AD發(fā)病中的作用及其與AD嚴(yán)重程度的相關(guān)性。在此基礎(chǔ)上,采用卵清蛋白(OVA)經(jīng)皮膚反復(fù)致敏誘導(dǎo)的AD小鼠模型,通過檢測AD小鼠皮損處IL-31mRNA、IL-31RA、OSMRβ及神經(jīng)營養(yǎng)素NT-4受體TrkB的表達(dá)及分布,探討IL-31及NT-4參與AD瘙癢的可能機(jī)制。 方法 1.符合Hanifin-Rajka診斷標(biāo)準(zhǔn)的AD患兒69例,采用SCORAD評分評估其疾病嚴(yán)重程度,ELISA方法測定患兒血清IL-31及NT-4水平。 2.使用OVA經(jīng)皮膚反復(fù)致敏誘導(dǎo)建立AD小鼠模型,采用實(shí)時(shí)定量RT-PCR法檢測AD小鼠皮損處IL-31mRNA表達(dá),同時(shí)ELISA法測定小鼠血清IL-31和NT-4水平。免疫組化法和蛋白印跡法(Western blot)檢測皮損處IL-31受體IL-31RA、OSMRβ及神經(jīng)營養(yǎng)素NT-4受體TrkB的分布及表達(dá)情況。 結(jié)果 1.本組69例AD患兒疾病嚴(yán)重程度評分SCORAD53.56±14.44,其中輕中度(SCORAD"f50)27例(39.1%),SCORAD39.78±8.92,重度(SCORAD50)42例(60.9%),SCORAD62.42±9.48。 2.AD患兒血清IL-3134.73±13.33pg/ml,顯著高于對照組(20.89±8.90pg/ml; p0.001),其中輕中度組28.52±13.00pg/ml,顯著高于對照組(p0.05),重度組38.72±12.08pg/ml,顯著高于對照組(p0.001)及輕中度組(p0.01)。 3.AD患兒血清NT-4中位數(shù)8.84ng/ml (1.55-45.75),顯著高于對照組(2.95ng/ml(0.82-21.12)；p0.01)；輕中度組10.61±9.17ng/ml及重度組14.73±11.90ng/ml,均顯著高于對照組(p0.05；p0.01)。 3.AD患兒SCORAD評分與血清IL-31水平呈正相關(guān)(r=0.557,p0.001)。AD患兒血清IL-31水平與血清NT-4水平呈正相關(guān)(r=0.515,p0.001)。在重度AD患兒中,血清NT-4水平與SCORAD呈正相關(guān)(r=0.327,p0.05)。 4.AD小鼠模型在致敏第6周,接觸部位明顯水腫,肥厚,皮損組織HE染色鏡下見AD小鼠表皮顆粒層增生,棘層肥厚,真皮層血管擴(kuò)張充血,膠原纖維顯著增生,并可見淋巴細(xì)胞、中性粒細(xì)胞及嗜酸性粒細(xì)胞等炎癥細(xì)胞浸潤,對照組基本正常。說明AD小鼠模型構(gòu)建成功。 5.AD小鼠皮損組織IL-31mRNA表達(dá)顯著增強(qiáng),是對照組的13.89倍(p0.01)。 6.AD小鼠血清IL-31水平(171.63±14.15pg/ml)顯著高于對照組152.15±14.19pg/ml,p0.05),AD小鼠血清NT-4水平(115.47±16.50pg/ml)較對照組(112.99±11.20pg/ml)差異無統(tǒng)計(jì)學(xué)意義(p0.05)。 7.AD小鼠皮損處可見IL-31RA在角質(zhì)形成細(xì)胞,毛囊細(xì)胞及巨噬細(xì)胞胞漿內(nèi)均呈強(qiáng)陽性表達(dá),OSMRβ主要表達(dá)于角質(zhì)形成細(xì)胞和毛囊細(xì)胞胞漿內(nèi),同時(shí)神經(jīng)營養(yǎng)素NT-4受體TrkB在角質(zhì)形成細(xì)胞胞漿及神經(jīng)纖維內(nèi)呈陽性表達(dá)。與對照組比較,AD小鼠皮損處IL-31RA蛋白表達(dá)顯著增強(qiáng)(0.78±0.43vs0.40±0.19；p0.05),TrkB蛋白表達(dá)增強(qiáng)(0.05±0.24vs0.03±0.16；p0.05),而OSMRP蛋白表達(dá)量無統(tǒng)計(jì)學(xué)差異(0.40±0.21vs0.28±0.17,p0.05)。 結(jié)論 1.AD患兒外周血IL-31水平明顯升高且與疾病嚴(yán)重程度呈正相關(guān)。AD小鼠血清IL-31水平與皮損處IL-31mRNA表達(dá)均顯著升高,證明IL-31與AD發(fā)生密切相關(guān),是反映AD疾病嚴(yán)重程度的重要指標(biāo)。 2.AD患兒外周血NT-4水平升高,同時(shí)血清IL-31與NT-4水平也呈正相關(guān)關(guān)系,說明神經(jīng)營養(yǎng)素NT-4可能在AD的發(fā)病中起重要作用。 3.在AD小鼠模型皮損處IL-31受體IL-31RA和OSMRP表達(dá)在角質(zhì)形成細(xì)胞胞漿內(nèi),神經(jīng)營養(yǎng)素NT-4受體TrkB在神經(jīng)纖維內(nèi)呈陽性表達(dá),同時(shí)IL-31RA和TrkB蛋白表達(dá)顯著增強(qiáng)。由此推測,IL-31可能經(jīng)由位于角質(zhì)形成細(xì)胞內(nèi)的IL-31RA/OSMRβ功能受體復(fù)合物,影響角質(zhì)形成細(xì)胞功能,促進(jìn)角質(zhì)形成細(xì)胞分泌NT-4,通過與神經(jīng)纖維內(nèi)的受體TrkB結(jié)合,參與AD瘙癢的病理生理過程。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R758.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 徐瑋;鄭敏;;神經(jīng)營養(yǎng)素與特應(yīng)性皮炎發(fā)病機(jī)制的進(jìn)展[J];國際皮膚性病學(xué)雜志;2006年03期

2 胡佳;張美華;畢志剛;陳文琦;董正邦;方晶;;卵清蛋白經(jīng)皮致敏誘發(fā)BALB/c小鼠特應(yīng)性皮炎樣病變的探討[J];臨床皮膚科雜志;2006年06期

3 顧恒,顏艷,陳],陳祥生,曹寧校,邵長庚,葉干運(yùn);我國特應(yīng)性皮炎流行病學(xué)調(diào)查[J];中華皮膚科雜志;2000年06期

4 顧恒,尤立平,劉永生,顏艷,陳昆;我國10城市學(xué)齡前兒童特應(yīng)性皮炎現(xiàn)況調(diào)查[J];中華皮膚科雜志;2004年01期

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