【摘要】：研究背景：腹膜纖維化迄今仍然是一個(gè)世界性的難題,是導(dǎo)致尿毒癥患者被迫中斷長(zhǎng)期腹膜透析的最主要因素。在非生理性腹膜透析液的長(zhǎng)期刺激下,就會(huì)導(dǎo)致腹膜纖維化的發(fā)生,其主要機(jī)理是腹膜間皮細(xì)胞從上皮細(xì)胞表型向肌纖維母細(xì)胞的轉(zhuǎn)分化,即上皮間質(zhì)轉(zhuǎn)化(EMT)過程的發(fā)生,由此啟動(dòng)細(xì)胞增殖和膠原等基質(zhì)的積聚等,主要信號(hào)轉(zhuǎn)導(dǎo)涉及到Smad通路、PI3-K/Akt/mTOR途徑等。雷帕霉素(rapamycin),通過與FK結(jié)合蛋白(FK-binding protein, FKBP)結(jié)合,阻斷哺乳動(dòng)物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)的表達(dá),進(jìn)而抑制細(xì)胞生長(zhǎng)與增殖。在腎、肝、心等多個(gè)臟器領(lǐng)域的研究中雷帕霉素在抗增殖和抗纖維化方面已經(jīng)顯示了重要作用。目前雷帕霉素在抗腹膜纖維化領(lǐng)域的作用也受到越來越多的重視。 目的：明確雷帕霉素對(duì)于腹膜透析模型大鼠腹膜纖維化的防治效果,闡明雷帕霉素對(duì)于腹膜透析模型大鼠腹膜纖維化的可能信號(hào)轉(zhuǎn)導(dǎo)分子機(jī)制。 實(shí)驗(yàn)方法：動(dòng)物層面,取45只正常SD大鼠,隨機(jī)分為5組：正常組,生理鹽水組,高糖組,低濃度雷帕治療組,高濃度雷帕治療組,6周后處死,評(píng)估腹膜功能，，取腹壁組織,行HE及α-SMA、TGF-β的免疫組化染色,分析各組腹壁纖維化情況；細(xì)胞層面,取正常SD大鼠腹膜,原代培養(yǎng)腹膜間皮細(xì)胞,取第3-4代細(xì)胞,予以不同濃度高糖(30,60,120mM)干預(yù),及不同濃度雷帕霉素(’10,100nM)干預(yù),作細(xì)胞劃痕實(shí)驗(yàn),同時(shí)于處理48h后提取蛋白,分析高糖對(duì)α-SMA及E-cadherin的變化,以及雷帕霉素對(duì)腹膜細(xì)胞EMT的影響。 結(jié)果：動(dòng)物層面：高糖組大鼠的腹膜功能相較正常組及生理鹽水組明顯下降,低濃度雷帕及高濃度雷帕均顯著改善腹膜功能(P0.05)；HE染色及α-SMA、TGF-β免疫組化顯示,高糖組呈現(xiàn)腹膜纖維化狀態(tài),低濃度雷帕及高濃度雷帕均顯著改善腹膜纖維化狀態(tài),且高濃度相較低濃度效果更明顯。細(xì)胞層面：高糖處理后細(xì)胞呈現(xiàn)EMT改變,表現(xiàn)為：α-SMA的表達(dá)量相較正常組明顯升高,E-cadherin的表達(dá)量明顯下降,遷移率明顯增加,且呈濃度相關(guān)性(均P0.05)。雷帕明顯改善EMT表現(xiàn),且呈濃度相關(guān)性(均P0.05)。 結(jié)論：通過動(dòng)物層面及細(xì)胞層面驗(yàn)證了雷帕霉素對(duì)高糖引起的腹膜纖維化有明顯的防治作用,在有效濃度范圍內(nèi),其防治效果與劑量呈一定的正相關(guān)。驗(yàn)證了EMT在腹膜纖維化過程中的作用。雷帕霉素抑制EMT的機(jī)制主要表現(xiàn)為：抑制腹膜間皮細(xì)胞遷移,減少TGF-β的分泌,抑制成纖維細(xì)胞的形成,抑制細(xì)胞外基質(zhì)形成。
[Abstract]:Background: peritoneal fibrosis is still a worldwide problem and the most important factor leading to the forced interruption of long-term peritoneal dialysis in uremic patients. The long-term stimulation of non-physiological peritoneal dialysate will lead to peritoneal fibrosis. The main mechanism of peritoneal fibrosis is the transformation of peritoneal mesothelial cells from epithelial cells phenotype to myofibroblasts, that is, the process of epithelial mesenchymal transformation into (EMT). The main signal transduction involved in the Smad pathway, such as PI3-K / Akt / mTOR pathway. Rapamycin (rapamycin), inhibits cell growth and proliferation by blocking the expression of rapamycin target (mammalian target of rapamycin, mTOR) by binding to FK binding protein (FK-binding protein, FKBP). Rapamycin has been shown to play an important role in anti-proliferation and anti-fibrosis in kidney, liver and heart. At present, the role of rapamycin in the field of anti-peritoneal fibrosis has been paid more and more attention. Aim: to investigate the preventive and therapeutic effects of rapamycin on peritoneal fibrosis in peritoneal dialysis rats and to elucidate the possible signal transduction molecular mechanism of rapamycin on peritoneal fibrosis in peritoneal dialysis rats. Methods: at animal level, 45 normal SD rats were randomly divided into 5 groups: normal group, normal saline group, high glucose group, low concentration rapa treatment group, high concentration rapa treatment group and high concentration rapa treatment group after 6 weeks, the peritoneal function was evaluated and the abdominal wall tissue was taken. HE and 偽 -SMA-TGF- 尾 immunohistochemical staining were used to analyze the situation of abdominal wall fibrosis in each group. At the cell level, peritoneal mesothelial cells of normal SD rats were taken out, primary peritoneal mesothelial cells were cultured, 3-4 passage cells were taken and treated with different concentrations of high glucose (30 ~ 60120mm). And different concentrations of rapamycin (10100nM) were used for cell scratch test. After 48 h treatment, protein was extracted to analyze the changes of 偽 -SMA and E-cadherin and the effect of rapamycin on EMT of peritoneal cells. Results: at animal level, the peritoneal function of rats in high glucose group was significantly lower than that in normal group and saline group. The peritoneal function of rats in low concentration and high concentration of rapa improved significantly (P0.05) and 偽 -SMA-TGF- 尾 immunohistochemical staining. In high glucose group, peritoneal fibrosis was observed, and low and high concentrations of rapa significantly improved the state of peritoneal fibrosis, and the effect of high concentration phase was more obvious than that of low concentration. Cell level: after high glucose treatment cells showed EMT changes. The expression of 偽 -SMA was significantly increased compared with the normal group the expression of E-cadherin decreased significantly mobility increased significantly and showed a concentration correlation (P0.05). Rapa significantly improved EMT performance, and showed a concentration correlation (P0.05). Conclusion: rapamycin has obvious preventive and therapeutic effect on peritoneal fibrosis induced by high glucose by animal and cell level. In the range of effective concentration, the preventive and therapeutic effects of rapamycin are positively correlated with the dose. The role of EMT in the process of peritoneal fibrosis was verified. The mechanism of rapamycin inhibiting EMT is that it inhibits the migration of peritoneal mesothelial cells, reduces the secretion of TGF- 尾, inhibits the formation of fibroblasts and inhibits the formation of extracellular matrix.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.5;R656.41

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 許麗艷,李恩民,蔡唯佳,沈忠英,曾毅;NGAL基因5’側(cè)翼區(qū)轉(zhuǎn)錄調(diào)控元件螢火蟲熒光素酶報(bào)告基因表達(dá)載體的構(gòu)建與鑒定[J];癌變.畸變.突變;2004年01期

2 張建國(guó),丁涵露,任姜汶,高聞達(dá);NGAL在急性藥物相關(guān)性間質(zhì)性腎炎中的表達(dá)及其意義[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2005年20期

3 陳新杰;吳保平;;Peutz-Jeghers綜合征錯(cuò)構(gòu)瘤性息肉APC,NGAL,COX-2基因表達(dá)及意義[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2007年01期

4 吳琳;王慶華;;急性腎損傷的早期標(biāo)志物NAGL[J];福州總醫(yī)院學(xué)報(bào);2010年02期

5 呂長(zhǎng)剛;齊鳳杰;;NGAL和MMP-9蛋白在浸潤(rùn)性乳腺癌中的表達(dá)及臨床意義[J];廣東醫(yī)學(xué);2011年10期

6 何小潔;楊毅華;陳慎仁;;NGAL與糖尿病腎病[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2008年09期

7 陳林興;張麗玲;黃華;陳慎仁;;NGAL與慢性粒細(xì)胞性白血病[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2010年01期

8 黃華;陳林興;陳慎仁;王君;;NGAL在慢性粒細(xì)胞性白血病中的表達(dá)及其診斷意義[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2010年04期

9 鄒育海;鄭興;;NGAL/24p3在心血管疾病中的價(jià)值[J];國(guó)際心血管病雜志;2009年01期

10 梁園園;葉建榮;馬海平;鄭宏;;先天性心臟病伴肺動(dòng)脈高壓患兒圍術(shù)期中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白水平的變化[J];中國(guó)當(dāng)代兒科雜志;2013年10期

相關(guān)會(huì)議論文 前1條

1 張秀峰;楊關(guān)根;廖秀軍;;NGAL與結(jié)直腸腫瘤[A];2011年浙江省肛腸外科學(xué)術(shù)大會(huì)暨結(jié)直腸肛門疾病診治新進(jìn)展學(xué)習(xí)班論文匯編[C];2011年

相關(guān)博士學(xué)位論文 前10條

1 毛山山;NGAL-NGALR在腎小球腎炎中的表達(dá)及其機(jī)制的實(shí)驗(yàn)研究[D];復(fù)旦大學(xué);2011年

2 肖銳;中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白（NGAL）在呼吸機(jī)相關(guān)性肺損傷小鼠模型中表達(dá)的研究[D];廣州醫(yī)學(xué)院;2010年

3 許麗艷;NGAL基因在食管癌細(xì)胞中的表達(dá)調(diào)控機(jī)制[D];北京工業(yè)大學(xué);2006年

4 蔡林君;HNL/NGAL用于急性腎損傷早期診斷及其免疫學(xué)測(cè)定方法研究[D];吉林大學(xué);2009年

5 車志宏;代謝綜合征相關(guān)新因子的探討[D];中南大學(xué);2008年

6 張丕顯;NGAL蛋白的重組表達(dá)與純化及其對(duì)食管癌細(xì)胞的體外誘導(dǎo)作用研究[D];汕頭大學(xué);2009年

7 桂耀松;mTOR信號(hào)通路參與肺纖維化發(fā)病的機(jī)制研究[D];北京協(xié)和醫(yī)學(xué)院;2013年

8 任發(fā)亮;西羅莫司誘導(dǎo)自噬對(duì)UVB輻照下HaCaT細(xì)胞增殖和凋亡的初步影響[D];北京協(xié)和醫(yī)學(xué)院;2013年

9 魏偉;大鼠肝移植慢性排斥反應(yīng)的蛋白組學(xué)研究[D];福建醫(yī)科大學(xué);2013年

10 宮晨;LY294002對(duì)人骨肉瘤類腫瘤干細(xì)胞的影響及其作用機(jī)制[D];華中科技大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 代巖;NGAL和MMP-10在非小細(xì)胞肺癌中的表達(dá)及臨床意義[D];鄭州大學(xué);2010年

2 馮金;2型糖尿病患者尿液NGAL與微量白蛋白尿的相關(guān)性研究[D];中國(guó)醫(yī)科大學(xué);2010年

3 張余黃;急性胰腺炎患者早期脂質(zhì)運(yùn)載蛋白-2的變化及對(duì)預(yù)后判斷的意義研究[D];瀘州醫(yī)學(xué)院;2011年

4 王卓;NGAL/Lcn2在感染幽門螺桿菌胃黏膜中的表達(dá)及臨床意義[D];蘭州大學(xué);2011年

5 趙璇;2型糖尿病患者血清NGAL水平與早期糖尿病腎病的關(guān)系[D];重慶醫(yī)科大學(xué);2011年

6 熊火梅;人NGAL原核表達(dá)及多克隆抗體的制備[D];南昌大學(xué);2011年

7 陳茜;Lipocalin-2與妊娠期糖尿病并發(fā)子癇前期相關(guān)性研究[D];廣州醫(yī)學(xué)院;2011年

8 鄔葉紅;尿中性粒細(xì)胞明膠酶相關(guān)性脂質(zhì)運(yùn)載蛋白對(duì)早期檢測(cè)SLE患者腎臟受損的臨床意義[D];復(fù)旦大學(xué);2011年

9 韓溟;酵母雙雜交系統(tǒng)的建立及NGAL相互作用蛋白的篩選與初步鑒定[D];汕頭大學(xué);2003年

10 劉志榮;大腸癌組織、腺瘤組織和正常黏膜組織的基因表達(dá)譜分析[D];山西醫(yī)科大學(xué);2006年

本文編號(hào)：2207707