本文選題：肺纖維化 + 環(huán)氧-二十碳三烯酸��； 參考：《中南大學(xué)》2012年碩士論文

【摘要】：肺纖維化(pulmonary fibrosis, PF)是由多種原因引起的慢性肺部疾病,其病理過(guò)程表現(xiàn)為肺組織損傷修復(fù)失調(diào)、細(xì)胞外基質(zhì)重構(gòu)、膠原過(guò)度沉積,進(jìn)而導(dǎo)致肺泡結(jié)構(gòu)破壞,最終發(fā)生肺纖維化。目前肺纖維的發(fā)病機(jī)制尚未完全闡明,療效不佳。當(dāng)細(xì)胞受到多種因素刺激時(shí),大量的花生四烯酸(arachidonic acid, AA)從細(xì)胞膜中分解釋放,并在細(xì)胞色素P450環(huán)氧化物酶(cytochrome P450, CYP)的作用下生成環(huán)氧-二十碳三烯酸(epoxyeicosatrienoic acids, EETs), EETs具有抗炎、舒張血管平滑肌、降低血壓、減輕心肌和大腦的缺血再灌注損傷以及抗凋亡等作用,但EETs能被可溶性環(huán)氧化物水解酶(soluble epoxide hydrolase, sEH)轉(zhuǎn)化為生物活性甚微的二羥基二十碳三烯酸(dihydroxyeicosatrienoic acids, DHET)。EETs對(duì)肺纖維化有無(wú)影響目前尚未見報(bào)道。本實(shí)驗(yàn)采用特異性sEH抑制劑TPPU抑制該酶的活性以增加胞內(nèi)EETs的含量,分別在整體水平和細(xì)胞水平探討EETs對(duì)肺纖維化的影響。 目的：觀察EETs對(duì)BLM致小鼠肺纖維化的影響以及對(duì)小鼠成纖維細(xì)胞增殖、分化和膠原合成的影響。 方法：(1)實(shí)驗(yàn)分為三組：正常組、BLM組、BLM+TPPU組,氣管注射BLM (10mg/kg)復(fù)制小鼠肺纖維化模型,通過(guò)灌胃給予小鼠TPPU(1mg/day/kg)以增加肺內(nèi)EETs的含量。采用HE和Masson染色觀察肺組織的形態(tài)學(xué)變化；Real-time PCR檢測(cè)小鼠肺組織膠原的表達(dá)； (2)采用MTT法觀察EETs對(duì)小鼠成纖維細(xì)胞(NIH3T3細(xì)胞株)增殖活性的影響；流式細(xì)胞術(shù)檢測(cè)EETs對(duì)小鼠成纖維細(xì)胞的細(xì)胞周期的影響；RT-PCR觀察EETs對(duì)TGF-β1誘導(dǎo)的小鼠成纖維細(xì)胞的α-SMA、Ⅰ型膠原及Ⅲ型膠原mRNA表達(dá)的影響。 結(jié)果： 1.動(dòng)物實(shí)驗(yàn) (1)小鼠肺組織形態(tài)學(xué)改變：HE染色顯示BLM組第21天肺纖維化病灶形成,TPPU+BLM組肺纖維化較BLM組顯著改善；Masson染色顯示BLM組第21天膠原大量沉積,TPPU+BLM組較BLM組顯著改善(P0.05)； (2) RT-PCR檢測(cè)顯示：與正常組相比,BLM組Ⅰ型膠原mRNA的表達(dá)顯著升高(P0.05)；與BLM組相比,TPPU+BLM組Ⅰ型膠原mRNA的表達(dá)顯著降低(P0.05)。 2.細(xì)胞實(shí)驗(yàn) (1)MTT法結(jié)果顯示：TPPU處理后呈時(shí)間依賴性抑制成纖維細(xì)胞的增殖(P0.01)；并呈劑量相關(guān)性(0.1μM,1μM,10μM)抑制成纖維細(xì)胞的增殖(P0.05)； (2)流式細(xì)胞術(shù)結(jié)果顯示：與正常組(17.24±2.12)相比,TGF-β1組(24.5±1.05)%成纖維細(xì)胞S期比例顯著增加(P0.05)；與DMSO+TGF-β1組(25.55±2.6)%相比,TPPU(1μM)+TGF-β1組(20.37±2.6)%和TPPU(10μM)+TGF-β1組(19.36±0.13)%成纖維細(xì)胞S期比例均顯著降低(P0.05)； (3)RT-PCR檢測(cè)結(jié)果顯示：與正常組相比,TGF-β1(?)應(yīng)激后成纖維細(xì)胞α-SMA mRNA的表達(dá)顯著升高(P0.05)；TPPU預(yù)處理組成纖維細(xì)胞α-SMA mRNA的表達(dá)顯著降低(P0.05)； (4)RT-PCR檢測(cè)結(jié)果顯示：與正常組相比,TGF-β1應(yīng)激成纖維細(xì)胞后可引起Ⅰ、Ⅲ型膠原mRNA的表達(dá)顯著增加(P0.05),TPPU預(yù)處理組可顯著降低成纖維細(xì)胞Ⅰ、Ⅲ型膠原mRNA的表達(dá)(P0.05)。 結(jié)論： 1.EETs可抑制博來(lái)霉素誘導(dǎo)的小鼠肺纖維化； 2.EETS可抑制成纖維細(xì)胞的增殖、分化及膠原合成。
[Abstract]:Pulmonary fibrosis ( PF ) is a chronic pulmonary disease caused by a variety of reasons . Its pathological process is lung tissue damage repair disorder , extracellular matrix remodeling , collagen overdeposition , which leads to destruction of alveolar structure and eventual pulmonary fibrosis . The effect of EETs on pulmonary fibrosis was not reported . The effect of EETs on pulmonary fibrosis was investigated by using specific sEH inhibitor TPPU to inhibit the activity of the enzyme to increase intracellular EETs .

Objective : To observe the effect of EETs on pulmonary fibrosis in mice and the effect of EETs on proliferation , differentiation and collagen synthesis of mouse fibroblast .

Methods : ( 1 ) The experiment was divided into three groups : normal group , blm group , blm + tppu group , intratracheal injection of blm ( 10 mg / kg ) to replicate lung fibrosis model of mice , and mice were given tppu ( 1 mg / day / kg ) by gavage to increase the content of eets in the lungs .
Real - time PCR was used to detect the expression of collagen in lung tissue of mice .


( 2 ) MTT assay was used to observe the effect of EETs on proliferation activity of mouse fibroblast ( NIH 3T3 cell line ) .
Effect of EETs on cell cycle of mouse fibroblasts by flow cytometry
The effects of EETs on the expression of 偽 - SMA , type鈪燾ollagen and type 鈪，

本文編號(hào)：1932714