本文關(guān)鍵詞： 特應(yīng)性皮炎 培土清心顆粒 小鼠特應(yīng)性皮炎模型 miR-146a　出處：《廣州中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的:1.檢測(cè)特應(yīng)性皮炎樣模型小鼠脾臟中Th1(CD4+IFN-Y+)、Th2(CD4+IL-4+)細(xì)胞和小鼠皮損中mi R-146a的表達(dá)情況。2.探討培土清心顆粒對(duì)特應(yīng)性皮炎樣模型小鼠脾臟中Th1、Th2細(xì)胞和小鼠皮損中miR-146a的表達(dá)的影響。3.檢測(cè)特應(yīng)性皮炎患者外周血單個(gè)核細(xì)胞(PBMC)中Th1、Th2細(xì)胞及血清中miR-146a的表達(dá)情況,探討miR-146a與特應(yīng)性皮炎嚴(yán)重程度(SCORAD)、IGA評(píng)分和中醫(yī)證型之間的相關(guān)性。方法:1.實(shí)驗(yàn)研究雄性C57BL/6小鼠隨機(jī)分為空白組、模型組、培土清心顆粒組,2,4-二硝基氟苯(DNFB)誘導(dǎo)建立特應(yīng)性皮炎模型,造模前24小時(shí)用剃毛刀剃除小鼠背部毛,并用脫毛膏脫毛,面積大小約2cm×2cmm,模型組和培土清心顆粒組第一天用50ul丙酮及橄欖油(丙酮:橄欖油=4:1,丙酮、橄欖油對(duì)皮膚無(wú)損害)配制成的0.5%DNFB溶液外涂于小鼠背部皮膚,第5天起,右側(cè)耳部、背部分別使用20ul、50ul 0.2%DNFB溶液重復(fù)致敏。每隔3天1次(第5、8、11、14天)�？瞻捉M小鼠背部及耳部皮膚外涂同樣量的丙酮:橄欖油=4:1基質(zhì)。培土清心顆粒組從造模第一天開(kāi)始用藥,連續(xù)用藥14天。造模完成后,觀察各組小鼠背部、耳部皮損表現(xiàn)、耳部腫脹程度及皮膚病理改變。流式分析法檢測(cè)小鼠脾臟中Th1、Th2細(xì)胞的表達(dá),RT-PCR法檢測(cè)小鼠皮損中mi R-146a的表達(dá)。2.臨床研究納入符合Hanifin和RajikaAD診斷標(biāo)準(zhǔn)且研究者整體評(píng)價(jià)(IGA)評(píng)分≥3分的AD患者25例,正常人16例,參照特應(yīng)性皮炎嚴(yán)重度(SCORAD)評(píng)分對(duì)患者進(jìn)行評(píng)分;參照2013年特應(yīng)性皮炎中醫(yī)診療方案專家共識(shí)為AD患者辯證分型;檢測(cè)患者1gE表達(dá)及過(guò)敏原;流式分析法檢測(cè)特應(yīng)性皮炎外周血單個(gè)核細(xì)胞(PBMC)中Th1、Th2表達(dá);RT-PCR法檢測(cè)特應(yīng)性皮炎患者血清中miR-146a表達(dá);分析miR-146a表達(dá)與SCORAD、IGA評(píng)分的相關(guān)性;分析不同證型中miR-146a表達(dá)情況。結(jié)果:1.空白組小鼠背部、耳部皮膚正常,模型組小鼠背部、耳部皮膚增厚、粗糙、紅斑、脫屑、結(jié)痂,培土清心顆粒組小鼠背部、耳部皮膚輕度增厚、粗糙、淡紅斑、少許脫屑、結(jié)痂;空白組小鼠耳部無(wú)腫脹,模型組小鼠耳部腫脹明顯,培土清心顆粒組小鼠耳部輕度腫脹,三組兩兩相比均具有統(tǒng)計(jì)學(xué)意義;空白組皮膚組織病理無(wú)明顯異常;模型組皮膚組織病理表現(xiàn)為表皮角化不全、角化過(guò)度、海綿水腫、棘層增厚,真皮內(nèi)大量淋巴細(xì)胞浸潤(rùn);培土清心顆粒組皮膚組織病理表現(xiàn)為表皮輕度角化不全、角化過(guò)度、海綿水腫,棘層輕度增厚,真皮內(nèi)少量淋巴細(xì)胞浸潤(rùn)。2.模型組、培土清心顆粒組Th1均較空白組降低,但差異沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05);模型組Th2較空白組、培土清心顆粒組升高,差異均有統(tǒng)計(jì)學(xué)意義(P0.01);培土清心顆粒組Th2較空白組升高,差異沒(méi)有統(tǒng)計(jì)學(xué)意義(P＞0.05);模型組Th1/Th2較空白組降低,差異有統(tǒng)計(jì)學(xué)意義(P0.01);模型組Th1/Th2較培土清心顆粒組降低,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05);培土清心顆粒組Th1/Th2較空白組降低,差異沒(méi)有統(tǒng)計(jì)學(xué)意義(P＞0.05)。3.模型組小鼠皮膚miR-146a表達(dá)較空白組小鼠高,差異具有統(tǒng)計(jì)學(xué)意義(P0.01);培土清心顆粒組小鼠皮膚miR-146a表達(dá)較空白組小鼠高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);培土清心顆粒組小鼠皮膚miR-146a表達(dá)較模型組降低,差異沒(méi)有統(tǒng)計(jì)學(xué)意義(P＞0.05)。4.25例特應(yīng)性皮炎(AD)患者中,6例患者是心脾積熱證,12例患者是心火脾虛證,4例患者是脾虛蘊(yùn)濕證,3例患者是血虛風(fēng)燥證,心火脾虛證患者miR-146a表達(dá)水平最高,心脾積熱證患者次之,脾虛蘊(yùn)濕證患者再次之,血虛風(fēng)燥證患者最低,但各組差異均沒(méi)有統(tǒng)計(jì)學(xué)意義(P＞0.05)。5.AD組Th1細(xì)胞較正常組降低,差異有統(tǒng)計(jì)學(xué)意義(P＜0.01);AD組Th2細(xì)胞較正常組升高,差異沒(méi)有統(tǒng)計(jì)學(xué)意義;AD組Th1/Th2較正常組降低,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。6.AD組血清中miR-146a表達(dá)較正常組高,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。miR-146a表達(dá)與SCORAD、IGA評(píng)分均沒(méi)有相關(guān)性。結(jié)論:1.2,4-二硝基氟苯(DNFB)誘導(dǎo)建立的C57BL/6小鼠特應(yīng)性皮炎模型是一個(gè)比較理想的用來(lái)研究特應(yīng)性皮炎發(fā)病機(jī)制的模型。2.培土清心顆粒有抑制特應(yīng)性皮炎模型炎癥反應(yīng)的作用,其作用機(jī)制可能通過(guò)調(diào)節(jié)Th1/Th2免疫失衡、抑制miR-146a表達(dá)等方面發(fā)揮作用。3.MiR-146a可能參與特應(yīng)性皮炎的發(fā)病過(guò)程。
[Abstract]:Objective: to detect 1. atopic dermatitis like model mice spleen Th1 (CD4+IFN-Y+), Th2 (CD4+IL-4+) cells and mouse MI in the lesions of R-146a expression of.2. Granule on hilling and clearing away heart fire atopic dermatitis like model mice spleen Th1,.3. detection on the expression of miR-146a Th2 cells and mouse skin lesions of atopic mononuclear cells in peripheral blood of patients with atopic dermatitis (PBMC) in Th1, the expression of miR-146a and Th2 cells in serum, to investigate the miR-146a and severity of atopic dermatitis (SCORAD), the correlation between IGA score and TCM syndrome. Methods: 1. experimental male C57BL/6 mice were randomly divided into control group, model group, group 2,4- two particles Qingxin hilling, 4-dinitrofluorobenzene (DNFB) induced atopic dermatitis model, before modeling 24 hours with razor to shave the mouse back hair, and use depilatory creams, the size of about 2cm * 2cmm, the model group and the formula of hilling and clearing away heart particles The first day of group with 50ul acetone and olive oil (=4:1 acetone: olive oil, acetone, olive oil on the skin without damage) 0.5%DNFB solution prepared onto the back skin of mice, the fifth day, the right ear and back respectively using 20ul 50ul 0.2%DNFB solution sensitized. Every 1 times 3 days (article 5,8,11,14 the day with the same amount of acetone). The control mice were back and ear skin: olive oil =4:1 matrix. From the beginning of particle group earth Qingxin model of the first day, 14 days of continuous use. After modeling, the mice were observed back, ear skin lesions, ear swelling and skin pathological changes of flow cytometric analysis. Detection of murine splenic Th1 cells, expression of Th2,.2. expression and clinical study on detection of mouse skin lesions in MI RT-PCR R-146a with Hanifin and RajikaAD into the overall evaluation of diagnostic criteria and Research (IGA) score more than 3 points in 25 patients with AD, 16 cases of normal people, According to the severity of atopic dermatitis (SCORAD) scores of the patients were scored according to 2013; atopic dermatitis expert consensus scheme of TCM diagnosis and treatment for patients with AD syndrome differentiation; the expression of 1gE and allergen detection were analyzed by flow cytometry; atopic dermatitis in peripheral blood mononuclear cells (PBMC) in Th1, Th2 expression; RT-PCR method was used to detect the expression of serum miR-146a in patients with atopic dermatitis; the expression of miR-146a and SCORAD, the correlation of IGA score; miR-146a expression analysis of different syndrome types. Results: 1. control group mice back, normal mice ear skin, back, ear skin thickening, rough, erythema, desquamation, crusting, "the back of the mice ear Qingxin granules, mild skin thickening, rough, pale little erythema, desquamation, crusting; ear swelling of mice in blank control group, model group, mouse ear swelling significantly, granule group hilling and clearing away heart fire mouse ear swelling, three The 22 groups were statistically significant compared with the blank group; skin tissue pathology showed no abnormalities; pathological skin tissue model showed epidermal hyperkeratosis, parakeratosis, acanthosis, spongiosis, dermal lymphocytic infiltration; skin tissue particles Qingxin hilling pathology showed mild epidermal hyperkeratosis and parakeratosis. The spongy, spinous layer thickened slightly, a small amount of dermal infiltration of lymphocytes in.2. model group and Th1 group were particles of hilling and clearing away heart fire decreased compared with the normal group, but the difference was not statistically significant (P0.05); Th2 in the model group than that in blank group, particles of hilling and clearing away heart fire group increased, the differences were statistically significant (P0.01); group Th2 particles Qingxin hilling compared with the blank group increased, the difference was not statistically significant (P > 0.05); model group Th1/Th2 decreased compared with the normal group, the difference was statistically significant (P0.01); model group Th1/Th2 is reducing hilling and clearing away heart fire particle group, difference There was statistical significance (P < 0.05); group Th1/Th2 particles of hilling and clearing away heart fire decreased compared with the normal group, the difference was not statistically significant (P > 0.05).3. model mice skin miR-146a expression compared with control group mice, the difference was statistically significant (P0.01); group miR-146a mouse skin particles Qingxin hilling expression compared to the control group of mice with high. Statistically significant difference (P0.05); the expression of miR-146a in mouse skin hilling and clearing away heart fire particle group lower than the model group, the difference was not statistically significant (P > 0.05).4.25 patients with atopic dermatitis (AD) patients, 6 cases of heart spleen heat retention syndrome, 12 cases of patients with firelight spleen deficiency, 4 patients is spleen deficiency in 3 patients with dampness, blood deficiency and wind dryness, heart spleen deficiency patients the expression level of miR-146a was highest, accumulation of heart and spleen syndrome of spleen deficiency in dampness again, with blood deficiency and wind dryness is lowest, but the difference was not statistically significant (P > 0 .05).5.AD in Th1 cells were lower than those in the normal group, the difference was statistically significant (P < 0.01); group AD Th2 cells than in the normal group, the difference was not statistically significant; AD group Th1/Th2 decreased compared with the normal group, the difference was statistically significant (P < 0.05) the expression of miR-146a in serum of group.6.AD was higher than that in normal group, have statistically significant difference (P < 0.05) the expression of.MiR-146a and SCORAD, IGA scores were not correlated. Conclusion: 1.2,4- two 4-dinitrofluorobenzene (DNFB) induced C57BL/6 mice atopic dermatitis model is an ideal model to study.2. atopic dermatitis incidence mechanism of hilling and clearing away heart fire particles can inhibit the atopic dermatitis model of inflammatory reaction, and its mechanism may be through regulating the immune imbalance of Th1/Th2, inhibition of miR-146a expression plays a role of.3.MiR-146a may be involved in the pathogenesis of atopic dermatitis.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R758.2;R-332

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