發(fā)布時間：2018-04-11 17:52

  本文選題：系統(tǒng)性紅斑狼瘡 + 狼瘡性腎炎��； 參考：《上海交通大學(xué)》2015年博士論文

【摘要】：【目的】系統(tǒng)性紅斑狼瘡(SLE)是由于機體免疫耐受缺失而引起的自身免疫性疾病,通常伴隨免疫復(fù)合物性腎小球腎炎,狼瘡性腎炎(LN)是其主要并發(fā)癥,嚴(yán)重影響患者預(yù)后。循環(huán)micro RNA(如血清micro RNA)在臨床疾病診斷中發(fā)揮重要作用,可以作為潛在的診斷標(biāo)記物。本文擬通過:1.microarray技術(shù)分析處于不同進展階段的LN患者血清mi RNA的變化。2.探討循環(huán)mi RNA對于LN的診斷價值及與LN臨床參數(shù)相關(guān)性。3.從分子生物學(xué)機制探討異常表達的循環(huán)mi RNA在LN發(fā)生發(fā)展中的作用�！痉椒ā�1.本研究一共囊括96份血清標(biāo)本,60例LN患者和36名健康對照組。挑選出其中性別、年齡匹配的12份血清標(biāo)本用于循環(huán)mi RNA表達譜篩選,包括早期LN患者4名(CKD分期1-3期),晚期LN患者4名(CKD分期4-5期)及健康體檢者4名。使用mi RNA PCR芯片檢測LN患者血清中372個mi RNA變化。2.剩余52名LN患者,包括40名早期LN患者、12名晚期LN患者及32名年齡、性別無差異的健康對照血清樣本用于驗證mi RNA表達譜篩選結(jié)果。200μL血清用于抽提總RNA,最后溶解于12μL無RNA酶水,Nanodrop 2000檢測血清RNA抽提濃度;使用tagman莖-環(huán)引物探針real-time PCR檢測差異循環(huán)mi RNA的相對表達量。分析循環(huán)mi RNA與LN患者臨床參數(shù)相關(guān)性,受試者工作曲線(ROC)及曲線下面積(AUC)分析循環(huán)mi RNA對疾病診斷的價值。3.腎小管上皮細(xì)胞(HK-2)分別轉(zhuǎn)染mi R-130b-3p mimics、inhibitor及各自相應(yīng)的對照,并予以TGF-β1(10ng/ml)刺激。實時定量PCR和westen blot檢測轉(zhuǎn)染72h后細(xì)胞Ⅰ型膠原、Ⅳ型膠原、E鈣連接素(E-cad)、α-平滑肌肌動蛋白(α-SMA)轉(zhuǎn)錄水平變化,westen blot檢測E-cad、α-SMA蛋白水平變化;檢測靶基因ERBB2IP及PPARγ的蛋白在轉(zhuǎn)染mimics后水平變化。構(gòu)建含有野生型ERBB2IP及PPARγ3’-UTR及突變的PPARγ3’-UTR雙熒光素報告酶載體,分別予以對照mimic、突變mimic及正常mimic共轉(zhuǎn)染,48h后檢測熒光活性�！窘Y(jié)果】1.與對照組相比,早期LN患者5個mi RNA表達下降倍數(shù)大于2,33個mi RNA上升倍數(shù)大于2。其中7個表達上升的mi RNA差異具有統(tǒng)計學(xué)意義;分別是:mi R-1233-3p(P=0.019),mi R-130b-3p(P=0.021),mi R-18a-3p(P=0.021),mi R-628-3p(P=0.023),mi R-1260b(P=0.030),mi R-1539(P=0.041)和mi R-378e(P=0.047),晚期LN組與健康對照組比較,共75個循環(huán)mi RNA表達下調(diào)大于2倍,其中P值小于0.05的mi RNA有21個;晚期LN組循環(huán)與早期LN組比較,共107個循環(huán)mi RNA表達下調(diào),其中53個mi RNA其表達下調(diào)具有統(tǒng)計學(xué)意義(P0.05)。對照組,早期LN患者、晚期LN患者RNA濃度依次為30.8(28.0,35.0)ng/μL、30.5(25.4,33.1)ng/μL、16.4(14.9,19.3)ng/μL,與對照組和早期LN組相比較,晚期LN患者血清總RNA濃度減少,其差異具有統(tǒng)計學(xué)意義(P0.01)。2.早期LN組患者與對照組比較,血清中存在mi R-130b-3p升高[IQR 16.2(8.7,42.7)vs 9.6(4.8,17.4),p0.01)],其差異具有統(tǒng)計學(xué)意義(P0.01),循環(huán)mi R-130b-3p對于區(qū)分LN患者與正常對照均具有一定價值價值,ROC曲線下面積(AUC)=0.683±0.062(95%CI=0.561-0.805,P0.01),最優(yōu)敏感度和特異度分別為35.05%和96.8%;驗證組顯示早期LN患者血清mi R-1233-3p雖然高于健康對照組患者[IQR 10.4(6.4,16.9)vs 8.7(3.8,16.9)],但其統(tǒng)計學(xué)差異未見明顯意義(P0.05);相關(guān)性分析顯示,循環(huán)mi R-130b-3p的相對表達量與LN患者疾病活動性各指標(biāo),如SLE疾病活動性評分(SLEDAI評分)、血清C3、C4、ds DNA、C反應(yīng)蛋白(CRP)、紅細(xì)胞沉降率(ESR)等無明顯相關(guān)性(P0.05),與24小時尿蛋白、慢性腎臟活動指數(shù)(CI)、甘油三酯(TG)呈正相關(guān)(P0.05)。3.正常HK-2細(xì)胞在轉(zhuǎn)染mi R-130b-3p mimic后,Ⅰ型膠原、Ⅳ型膠原、E鈣連接素(E-cad)、α-平滑肌肌動蛋白(α-SMA)表達并沒有顯著差異;但在TGF-β1刺激時,mi R-130b-3p的轉(zhuǎn)染可以增加小管上皮細(xì)胞Ⅰ型膠原、Ⅳ型膠原、α-平滑肌肌動蛋白(α-SMA)的m RNA和蛋白表達水平,并下調(diào)E-cad的表達,差異具有統(tǒng)計學(xué)意義(P0.05);相反的作用見于轉(zhuǎn)染mi R-130b-3p inhibitor;轉(zhuǎn)染mi R-130b-3p mimics無論伴或者不伴有TGF-β1(10ng/ml)刺激的情況下,與對照組相比,mi R-130b-3p mimics可以顯著下調(diào)PPAR-γ或者ERBB2IP蛋白表達水平(P0.05);mi R-130b-3p mimics與構(gòu)建ERBB2IP基因的3'UTR及突變的熒光素酶報告質(zhì)粒共轉(zhuǎn)染工具細(xì)胞293T細(xì)胞后,與NC和mut-mi R-130b-3p組相比,wt-mi R-130b-3p mimics與質(zhì)粒共轉(zhuǎn)染使海腎熒光素酶活性下降,海腎熒光素酶與螢火蟲熒光素酶活性之比下降(P0.01),而NC-mimics及mut-mi R-130b-3p對質(zhì)粒的作用無顯著性差異(P0.05);mi R-130b-3p mimics使wt-PPAR-γ-3’-UTR海腎熒光素酶與螢火蟲熒光素酶活性之比下降(P0.01),NC mimic及mut-PPAR-γ無此作用�！窘Y(jié)論】LN腎炎患者存在血清mi RNA表達異常;對于嚴(yán)重腎功能受損患者,循環(huán)mi RNA存在整體下降;早期升高的循環(huán)mi R-130b-3p可能通過與PPAR-γ和ERBB2IP 3’-UTR直接結(jié)合而調(diào)控腎小管上皮細(xì)胞EMT作用,參與早期LN腎臟損害。
[Abstract]:[Objective] systemic lupus erythematosus (SLE) is caused by the lack of immune tolerance and autoimmune disease, often accompanied by immune complex glomerulonephritis, lupus nephritis (LN) is the main complication, seriously affect the prognosis of patients with RNA. Micro cycle (such as serum micro RNA) play an important role in the clinical the diagnosis of disease, can be used as a potential diagnostic marker. This paper proposed by: in the analysis of 1.microarray technology.2. LN RNA changes of serum MI in patients with different stages of progress on the role in the occurrence and development of LN in MI RNA mi RNA for cycle cycle the diagnostic value of LN and LN and the clinical parameters between.3. from molecular biology mechanism of abnormal expression. [method] this study included a total of 1. of 96 serum samples, 60 cases of LN patients and 36 healthy controls. The selected one of the gender, age matched 12 serum specimens for circulating mi The expression profile of RNA screening, including 4 patients with early LN (CKD stage 1-3), 4 patients with advanced LN (CKD stage 4-5) and 4 healthy controls. The remaining 52 patients with LN 372 mi RNA.2. to detect changes of serum LN in patients with MI RNA PCR chip, including 40 patients with early LN 12, late stage LN patients and 32 age-matched healthy controls, no gender difference in serum samples used to validate mi RNA expression.200 L screening results for serum total RNA extracted, finally dissolved in 12 L water 2000 Nanodrop without RNA enzyme, serum RNA extraction concentration; the relative expression of circulating mi RNA assay. The use of tagman stem loop primers. Real-time PCR analysis of circulating mi RNA with clinical parameters of patients with LN correlation receiver operating curve (ROC) and area under the curve (AUC) analysis of circulating mi RNA on the diagnosis value of.3. in renal tubular epithelial cells (HK-2) were transfected into mi R-130b-3p mimics, inhib Itor and the corresponding control, and be TGF- beta 1 (10ng/ml) stimulation. Real time quantitative PCR and westen blot detection after transfection of 72h cells of type I collagen, type IV collagen, E calcium ligatin (E-cad), alpha smooth muscle actin (alpha -SMA) transcription level changes, westen blot detection of E-cad, changes of alpha -SMA protein level; protein detection of target gene ERBB2IP and PPAR gamma changes in level mimics after transfection. Construct containing wild type ERBB2IP and PPAR Gamma 3 '-UTR and mutation of PPAR Gamma 3' -UTR dual luciferase reporter vector, respectively, compared with mimic, mimic mutation and normal mimic co transfection, fluorescence detection [48H activity. Results 1.] compared with the control group, 5 patients with early LN mi RNA decreased the expression of multiple mi RNA rose more than 2,33 times greater than 2. of which were statistically significant differences in expression of MI RNA increased 7; respectively is: Mi R-1233-3p (P=0.019), MI R-130b-3p (P=0.021), MI R-18a -3p(P=0.021),mi R-628-3p(P=0.023),mi R-1260b(P=0.030),mi R-1539(P=0.041)鍜宮i R-378e(P=0.047),鏅氭湡LN緇勪笌鍋ュ悍瀵圭収緇勬瘮杈，

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