發(fā)布時(shí)間：2018-01-08 17:03

  本文關(guān)鍵詞：三棱丸方對(duì)克羅恩病腸纖維化的影響及機(jī)制研究　出處：《南京中醫(yī)藥大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 克羅恩病 腸纖維化 三棱丸 TGF-β1/Smads 過(guò)氧化物酶體增殖物激活受體-γ

【摘要】：目的使用克羅恩病腸纖維化大鼠模型觀察三棱丸方對(duì)大鼠腸纖維化的影響,觀察三棱丸在轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)所誘導(dǎo)的腸上皮細(xì)胞(IEC-6)上皮間質(zhì)轉(zhuǎn)化(EMT)過(guò)程中對(duì)纖維化相關(guān)因子及PPAR-γ表達(dá)的影響,并探討三棱丸方通過(guò)TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)途徑干預(yù)腸纖維化的EMT的機(jī)制。方法 體內(nèi)實(shí)驗(yàn)部分:將48只SD大鼠隨機(jī)均分為空白組、模型組、羅格列酮對(duì)照組、三棱丸方高、中、低劑量治療組,組除空白組外,后五組采每周一次給予5%的三硝基苯磺酸(trinitrobenzene sulphonic acid TNBS),1Omg 于第 1 天,15mg 于第 8 天,20mg 于第 15 天,25mg于第22天,30mg于第29天,分別配成50%乙醇溶液0.8ml 一一次性灌腸,逐漸增量灌腸法誘導(dǎo)腸纖維化模型;從造模開(kāi)始,大鼠同時(shí)每日分別以10.Og/kg,5.0g/kg,2.5g/kg不同濃度的三棱丸方水煎劑灌胃,羅格列酮組以8mg/kg濃度羅格列酮灌胃,每天一次;模型組、正常組給予等量生理鹽水灌胃。期間觀察各組大鼠的一般情況,35天后收集結(jié)腸組織,用于病理學(xué)評(píng)價(jià),并檢測(cè)結(jié)腸組織中TGF-β1、E-cadherin、α-SMA、FN、CTGF及PPARy水平。體外實(shí)驗(yàn)部分:以TGF-β1誘導(dǎo)IEC-6細(xì)胞形成的EMT模型為研究對(duì)象,用Western blot法檢測(cè)高、中、低劑量的三棱丸方含藥血清及羅格列酮作用于1EC-6細(xì)胞后PPARγ、Smad2/3及pSmad3蛋白的表達(dá),RT-PCR法檢測(cè)E-cadherin、α-SMA的mRNA的表達(dá);免疫熒光法觀察三棱丸含藥血清及羅格列酮對(duì)PPARγ、pSmad3入核水平的影響,并檢測(cè)加入PPARγ拮抗劑GW9662后,上述相關(guān)指標(biāo)發(fā)生的變化。結(jié)果體內(nèi)實(shí)驗(yàn)研究:與模型組比較,三棱丸方中、高劑量組的疾病活動(dòng)指數(shù)(DAI)評(píng)分、結(jié)腸大體評(píng)分、組織學(xué)纖維化評(píng)分均明顯低于模型組(P0.05),Masson染色顯示三棱丸方高劑量組膠原纖維含量明顯低于模型組;E-cadherin、PPARγ含量均較模型組明顯增加(P0.05),TGF-[β1、FN、α-SMA水平均明顯降低(P0.05)。體外實(shí)驗(yàn)研究:與10%正常大鼠血清組比較,三棱丸含藥血清組能夠抑制α-SMA,促進(jìn)E-cadherin、PPARγ的mRNA的表達(dá);提高E-cadherin、PPAR-γ,降低pSmad3、α-SMA的蛋白水平;免疫熒光結(jié)果顯示三棱丸含藥血清組促進(jìn)PPARγ入核,抑制pSmad3的入核;加入PPARy拮抗劑GW9662后,逆轉(zhuǎn)了上述指標(biāo)的變化趨勢(shì)。結(jié)論三棱丸方能改善大鼠CD腸纖維化模型的纖維化程度,對(duì)IEC-6細(xì)胞的EMT有抑制的作用;其治療克羅恩病腸纖維化的機(jī)制可能是和激活PPAR-γ來(lái)抑制TGF-β1/Smads的信號(hào)通路相關(guān)。
[Abstract]:The purpose of the use of Crohn's disease intestinal fibrosis rat model to observe the effect of sanlengwan Decoction on intestinal fibrosis in rats, observe the sanlengwan in transforming growth factor beta 1 (TGF- beta 1) induced by intestinal epithelial cells (IEC-6) of epithelial mesenchymal transition (EMT) process of fibrosis related factors and PPAR- expression. And to explore the mechanism of TGF beta sanlengwan through 1/Smads signal transduction pathway intervention of intestinal fibrosis in vivo EMT. Methods: 48 SD rats were randomly divided into control group, model group, rosiglitazone group, Sanleng pill high, low dose treatment group, group except the blank group, after five group each week for 5% three trinitrobenzene sulfonic acid (trinitrobenzene sulphonic acid TNBS 1Omg), on the first day, 15mg on the eighth day, 20mg on the fifteenth day, 25mg on the twenty-second day, 30mg on the twenty-ninth day, respectively, with 50% 0.8ml ethanol solution of a disposable enema, gradually increase The amount of enema induced intestinal fibrosis model; from the start of the model, the rats of the day were 10.Og/kg, 5.0g/kg, 2.5g/kg different concentrations of sanlengwan Decoction orally, rosiglitazone group with 8mg/kg concentration of rosiglitazone orally, once a day; the model group and normal group were given equal volume of saline. During the period of observation the situation of the rats were collected 35 days after colon tissue for pathological evaluation, and detection of colonic tissue TGF- beta 1, E-cadherin, FN, alpha -SMA, CTGF and PPARy. In vitro experiment: TGF- beta 1 induced IEC-6 cells to form EMT model as the research object, using the Western blot assay. In the low dose of sanlengwan medicated serum and rosiglitazone on 1EC-6 cell by PPAR, expression of Smad2/3 and pSmad3 protein, E-cadherin RT-PCR detection method, the expression of alpha -SMA mRNA; immunofluorescence observation of serum and Roger sanlengwan containing columns Ketone of PPAR gamma, pSmad3 effect into the level of the nucleus, and detected by PPAR gamma antagonist GW9662, change the relevant indicators. The experimental results of in vitro: compared with model group, three pills in high dose group disease activity index (DAI) score, colonic general score, histological fibrosis scores were significantly lower than the model group (P0.05), Masson staining showed that the three pill high dose group collagen fiber content was significantly lower than the model group; E-cadherin, PPAR gamma content increased than model group (P0.05), TGF-[beta 1, FN, alpha -SMA levels were decreased significantly (P0.05). In vitro study: compared with 10% normal rat serum group, sanlengwan medicated serum can inhibit alpha -SMA, promote the expression of PPAR E-cadherin and mRNA E-cadherin gamma; improve, reduce pSmad3, PPAR- gamma, alpha -SMA protein level; immunofluorescence results showed sanlengwan containing serum promote PPAR gamma nuclear entry, suppression PSmad3 into the nucleus; adding PPARy antagonist, GW9662, reversed the trend of the index. The degree of fibrosis conclusion sanlengwan decoction can improve the intestinal fibrosis in rats with CD model, on IEC-6 cell EMT has inhibiting effect; the treatment of Crohn's disease and intestinal fibrosis mechanism may be the activation of signal transduction pathways to PPAR- gamma inhibition of TGF- beta 1/Smads.

【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R285.5

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