MiR-146a在特应性皮炎患者和动物模型中的表达及意义

发布时间：2018-01-21 23:15

本文关键词： 特应性皮炎培土清心颗粒小鼠特应性皮炎模型 miR-146a　出处：《广州中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:1.检测特应性皮炎样模型小鼠脾脏中Th1(CD4+IFN-Y+)、Th2(CD4+IL-4+)细胞和小鼠皮损中mi R-146a的表达情况。2.探讨培土清心颗粒对特应性皮炎样模型小鼠脾脏中Th1、Th2细胞和小鼠皮损中miR-146a的表达的影响。3.检测特应性皮炎患者外周血单个核细胞(PBMC)中Th1、Th2细胞及血清中miR-146a的表达情况,探讨miR-146a与特应性皮炎严重程度(SCORAD)、IGA评分和中医证型之间的相关性。方法:1.实验研究雄性C57BL/6小鼠随机分为空白组、模型组、培土清心颗粒组,2,4-二硝基氟苯(DNFB)诱导建立特应性皮炎模型,造模前24小时用剃毛刀剃除小鼠背部毛,并用脱毛膏脱毛,面积大小约2cm×2cmm,模型组和培土清心颗粒组第一天用50ul丙酮及橄榄油(丙酮:橄榄油=4:1,丙酮、橄榄油对皮肤无损害)配制成的0.5%DNFB溶液外涂于小鼠背部皮肤,第5天起,右侧耳部、背部分别使用20ul、50ul 0.2%DNFB溶液重复致敏。每隔3天1次(第5、8、11、14天)。空白组小鼠背部及耳部皮肤外涂同样量的丙酮:橄榄油=4:1基质。培土清心颗粒组从造模第一天开始用药,连续用药14天。造模完成后,观察各组小鼠背部、耳部皮损表现、耳部肿胀程度及皮肤病理改变。流式分析法检测小鼠脾脏中Th1、Th2细胞的表达,RT-PCR法检测小鼠皮损中mi R-146a的表达。2.临床研究纳入符合Hanifin和RajikaAD诊断标准且研究者整体评价(IGA)评分≥3分的AD患者25例,正常人16例,参照特应性皮炎严重度(SCORAD)评分对患者进行评分;参照2013年特应性皮炎中医诊疗方案专家共识为AD患者辩证分型;检测患者1gE表达及过敏原;流式分析法检测特应性皮炎外周血单个核细胞(PBMC)中Th1、Th2表达;RT-PCR法检测特应性皮炎患者血清中miR-146a表达;分析miR-146a表达与SCORAD、IGA评分的相关性;分析不同证型中miR-146a表达情况。结果:1.空白组小鼠背部、耳部皮肤正常,模型组小鼠背部、耳部皮肤增厚、粗糙、红斑、脱屑、结痂,培土清心颗粒组小鼠背部、耳部皮肤轻度增厚、粗糙、淡红斑、少许脱屑、结痂;空白组小鼠耳部无肿胀,模型组小鼠耳部肿胀明显,培土清心颗粒组小鼠耳部轻度肿胀,三组两两相比均具有统计学意义;空白组皮肤组织病理无明显异常;模型组皮肤组织病理表现为表皮角化不全、角化过度、海绵水肿、棘层增厚,真皮内大量淋巴细胞浸润;培土清心颗粒组皮肤组织病理表现为表皮轻度角化不全、角化过度、海绵水肿,棘层轻度增厚,真皮内少量淋巴细胞浸润。2.模型组、培土清心颗粒组Th1均较空白组降低,但差异没有统计学意义(P0.05);模型组Th2较空白组、培土清心颗粒组升高,差异均有统计学意义(P0.01);培土清心颗粒组Th2较空白组升高,差异没有统计学意义(P＞0.05);模型组Th1/Th2较空白组降低,差异有统计学意义(P0.01);模型组Th1/Th2较培土清心颗粒组降低,差异有统计学意义(P＜0.05);培土清心颗粒组Th1/Th2较空白组降低,差异没有统计学意义(P＞0.05)。3.模型组小鼠皮肤miR-146a表达较空白组小鼠高,差异具有统计学意义(P0.01);培土清心颗粒组小鼠皮肤miR-146a表达较空白组小鼠高,差异具有统计学意义(P0.05);培土清心颗粒组小鼠皮肤miR-146a表达较模型组降低,差异没有统计学意义(P＞0.05)。4.25例特应性皮炎(AD)患者中,6例患者是心脾积热证,12例患者是心火脾虚证,4例患者是脾虚蕴湿证,3例患者是血虚风燥证,心火脾虚证患者miR-146a表达水平最高,心脾积热证患者次之,脾虚蕴湿证患者再次之,血虚风燥证患者最低,但各组差异均没有统计学意义(P＞0.05)。5.AD组Th1细胞较正常组降低,差异有统计学意义(P＜0.01);AD组Th2细胞较正常组升高,差异没有统计学意义;AD组Th1/Th2较正常组降低,差异有统计学意义(P＜0.05)。6.AD组血清中miR-146a表达较正常组高,差异有统计学意义(P＜0.05)。miR-146a表达与SCORAD、IGA评分均没有相关性。结论:1.2,4-二硝基氟苯(DNFB)诱导建立的C57BL/6小鼠特应性皮炎模型是一个比较理想的用来研究特应性皮炎发病机制的模型。2.培土清心颗粒有抑制特应性皮炎模型炎症反应的作用,其作用机制可能通过调节Th1/Th2免疫失衡、抑制miR-146a表达等方面发挥作用。3.MiR-146a可能参与特应性皮炎的发病过程。
[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.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R758.2;R-332

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