發(fā)布時(shí)間：2018-01-29 02:48

  本文關(guān)鍵詞： microRNA 微囊泡 腎小管間質(zhì)纖維化 腎小管上皮細(xì)胞表型轉(zhuǎn)化 PTEN microRNA 微囊泡 成纖維細(xì)胞 凋亡 腎小管間質(zhì)纖維化 Bcl-2 促紅細(xì)胞生成素 microRNA 微囊泡 腎小管基底膜 組織纖溶酶原激活劑　出處：《南京醫(yī)科大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：無(wú)論原發(fā)病因如何,大多數(shù)慢性腎臟病最終將進(jìn)展至腎小管間質(zhì)纖維化。臨床上,腎小管間質(zhì)纖維化的病變通常呈現(xiàn)由局部到彌漫和不可逆的特征,且病程的進(jìn)展與原發(fā)病因無(wú)關(guān)。在梗阻性腎小管間質(zhì)纖維化的動(dòng)物模型中,腎小管上皮細(xì)胞的表型轉(zhuǎn)化和腎小管間質(zhì)纖維化的病變也表現(xiàn)為局灶起病和彌漫進(jìn)展,且病變加重的速率與梗阻壓力的增速無(wú)關(guān)。腎小管上皮細(xì)胞在腎小管間質(zhì)纖維化中發(fā)揮重要作用,本研究探討損傷的腎小管上皮細(xì)胞本身是否促進(jìn)了腎小管間質(zhì)纖維化的進(jìn)展。以單側(cè)輸尿管梗阻(UUO)建立腎小管間質(zhì)纖維化動(dòng)物模型和培養(yǎng)的近端腎小管上皮細(xì)胞(NRK-52E)系為研究對(duì)象,發(fā)現(xiàn)損傷的腎小管上皮細(xì)胞分泌的微囊泡(MV)能夠誘導(dǎo)腎小管上皮細(xì)胞發(fā)生表型轉(zhuǎn)化；腎小管上皮細(xì)胞損傷后,miR-21的表達(dá)顯著增加并被分泌到微囊泡中；微囊泡將具備生物學(xué)活性的外源性miR-21傳遞到腎小管上皮細(xì)胞后,通過(guò)抑制靶蛋白PTEN而增強(qiáng)Akt信號(hào)誘導(dǎo)腎小管上皮細(xì)胞表型轉(zhuǎn)化。本研究從微囊泡介導(dǎo)的細(xì)胞間miRNA傳遞的角度探討腎小管間質(zhì)纖維化進(jìn)展的機(jī)制,將有助于研發(fā)新的慢性腎臟病的治療靶點(diǎn)。腎纖維化以腎小管萎縮和細(xì)胞外基質(zhì)蓄積為特征,小管上皮細(xì)胞凋亡是腎小管萎縮和間質(zhì)纖維化的病因之一,然而,腎臟小管上皮細(xì)胞凋亡的調(diào)節(jié)機(jī)制仍不清楚。MicroRNA是一類(lèi)內(nèi)源性的非編碼小RNA,調(diào)節(jié)細(xì)胞的增殖分化代謝和死亡等過(guò)程,研究表明microRNA(miR)-34a調(diào)節(jié)細(xì)胞凋亡。MicroRNA不僅能夠調(diào)節(jié)自身細(xì)胞的靶基因表達(dá),還通過(guò)微囊泡傳遞來(lái)調(diào)節(jié)其他細(xì)胞的靶基因表達(dá)。本研究以梗阻性腎纖維化模型及培養(yǎng)的腎小管上皮細(xì)胞和腎間質(zhì)成纖維細(xì)胞為研究對(duì)象,探討包含miR-34a的微囊泡調(diào)控腎小管上皮細(xì)胞凋亡的機(jī)制。發(fā)現(xiàn)在梗阻的腎組織中,miR-34a的表達(dá)增高并主要分布在腎小管間質(zhì)細(xì)胞中。腎組織的微囊泡中miR-34a的表達(dá)在梗阻后顯著增高。TGF-β1處理上調(diào)了腎間質(zhì)成纖維細(xì)胞而非近端腎小管上皮中miR-34a的表達(dá)。腎間質(zhì)成纖維細(xì)胞分泌包含miR-34a的微囊泡,經(jīng)破損的小管基底膜傳遞到近端腎小管上皮細(xì)胞中,通過(guò)抑制Bcl-2的表達(dá)誘導(dǎo)細(xì)胞凋亡。本研究從包含microRNA的微囊泡介導(dǎo)細(xì)胞間通訊的角度,為認(rèn)識(shí)腎小管上皮細(xì)胞的凋亡提供新的分子機(jī)制,為解釋腎纖維化的發(fā)病機(jī)制提供新的理論依據(jù)。腎間質(zhì)纖維化是慢性腎臟病進(jìn)展的顯著特征,腎小管基底膜完整性的破壞在腎臟纖維化過(guò)程中發(fā)揮關(guān)鍵作用。組織纖溶酶原激活劑(tissue plasminogen activator, tPA)通過(guò)激活基質(zhì)金屬蛋白酶(matrix metalloproleinases, MMP)-9,介導(dǎo)腎小管基底膜的降解,是調(diào)節(jié)基底膜完整性的重要因素。tPA是miR-144的靶基因。注射促紅細(xì)胞生成素(erythropoietin, EPO)后,血清miR-144的水平顯著上升,血清中的miRNA可能包含在微囊泡中。有研究發(fā)現(xiàn),EPO能保護(hù)腎臟,延緩腎臟纖維化。本研究以單側(cè)輸尿管梗阻(UUO)的腎組織和腎間質(zhì)成纖維細(xì)胞為研究對(duì)象,探討EPO延緩腎小管間質(zhì)纖維化的機(jī)制。結(jié)果顯示,注射EPO能顯著減輕梗阻腎的小管基底膜完整性的破壞,梗阻腎組織中tPA的表達(dá)增高和MMP-9的活性增加得到顯著減輕。小鼠注射EPO后,血清微囊泡中miR-144的含量顯著增高,EPO小鼠血清的微囊泡能抑制培養(yǎng)的腎間質(zhì)成纖維細(xì)胞中tPA的表達(dá)和細(xì)胞培養(yǎng)液中MMP-9的活性,并顯著減輕TGF-β1誘導(dǎo)的腎間質(zhì)成纖維細(xì)胞中tPA表達(dá)和細(xì)胞培養(yǎng)液中MMP-9活化。EPO小鼠血清的微囊泡能減輕梗阻腎的小管基底膜完整性的破壞,緩解梗阻腎組織中tPA的表達(dá)增高和MMP-9的活性增加。調(diào)節(jié)miR-144的表達(dá)能負(fù)性調(diào)控腎間質(zhì)成纖維細(xì)胞中tPA的表達(dá)和MMP-9的活性。過(guò)表達(dá)miR-144能拮抗,而抑制miR-144則加重梗阻TGF-β1誘導(dǎo)的腎間質(zhì)成纖維細(xì)胞中tPA表達(dá)和細(xì)胞培養(yǎng)液中MMP-9活化。本研究提示EPO通過(guò)上調(diào)血清微囊泡中miR-144的含量,抑制梗阻腎組織的成纖維細(xì)胞中tPA的表達(dá)和MMP-9的活化,減輕小管基底膜完整性的破壞,從而延緩腎小管間質(zhì)纖維化的進(jìn)展。
[Abstract]:No matter how the primary cause of chronic kidney disease, most will eventually progress to renal tubulointerstitial fibrosis. Clinically, tubulointerstitial fibrosis lesions usually present from local to diffuse and non reversible, and the progress of the disease and the primary cause. In the animal model of fibrosis in obstructive renal tubule renal tubular epithelial cells, and phenotypic transformation of renal tubular interstitial fibrosis lesions as well as the progress of focal onset and diffuse, obstruction rate and pressure and aggravate the lesion of renal tubular epithelial cells. The growth rate has played an important role in renal tubulointerstitial fibrosis, to investigate the damage of renal tubular epithelial cell itself is to promote the progression of tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO) of renal tubule interstitial fibrosis animal model and proximal renal tubular epithelial cell culture system (NRK-52E). The object that microcapsule secretion of renal tubular epithelial cell injury of the bubble (MV) can induce the transdifferentiation of renal tubular epithelial cells; renal tubular epithelial cells after injury, the expression of miR-21 was significantly increased and was secreted into the vesicles; vesicles will have the biological activity of exogenous miR-21 transfer to renal tubular epithelial cells and through the inhibition of PTEN target protein and enhanced Akt signal induced tubular epithelial cell phenotype transformation. To investigate the mechanism of renal tubulointerstitial fibrosis in this study from vesicles mediated intercellular miRNA transfer point, there will be therapeutic targets help to develop new chronic kidney disease. Kidney fibrosis in renal tubular atrophy and the extracellular matrix accumulation characteristics, apoptosis of tubular epithelial cell is one of the causes of interstitial fibrosis, tubular atrophy, and however, regulation mechanism of apoptosis of renal tubular epithelial cells is still not clear.MicroR NA is a class of endogenous small non encoding RNA, the proliferation and differentiation of metabolism and regulation of cell death process, research shows that microRNA (miR) -34a gene expression regulation of cell apoptosis in.MicroRNA cells can not only regulate itself, but also through microvesicles targeted gene delivery to regulate the expression of other cells. In this study, renal tubular epithelial cells obstructive renal and renal fibrosis model and cultured stromal fibroblasts as the research object, to explore the mechanism of microcapsules containing miR-34a global regulation of apoptosis of renal tubular epithelial cells. Found in obstruction in renal tissue, the expression of miR-34a increased and mainly distributed in the renal tubular interstitial cells. The expression of renal tissue foam microcapsule miR-34a after obstruction was significantly higher in the.TGF- beta 1 treatment increased renal interstitial fibroblasts and the expression of miR-34a in the proximal tubular epithelium. Renal interstitial fibroblasts containing miR-34a Vesicles, the tubular basement membrane damage is transferred to the proximal renal tubular epithelial cells, induce cell apoptosis by inhibiting expression of Bcl-2. This study from the perspective of microcapsules containing microRNA vesicle mediated intercellular communication, provide a new molecular mechanism for understanding the apoptosis of renal tubular epithelial cells, and provide a new theoretical basis for to explain the pathogenesis of renal fibrosis. Renal interstitial fibrosis is a prominent feature of the progression of chronic kidney disease, the integrity of the tubular basement membrane damage plays a key role in the process of renal fibrosis. Tissue plasminogen activator (tissue plasminogen, activator, tPA) through the activation of matrix metalloproteinases (matrix, metalloproleinases, -9, MMP) mediated degradation the tubular basement membrane,.TPA is an important factor in regulating the integrity of basement membrane is the target gene of miR-144. Injection of erythropoietin (erythropoietin, EPO), serum mi The level of R-144 increased significantly, serum miRNA may be contained in the vesicles. Studies have found that EPO can protect the kidney, and delay renal fibrosis. In this study, unilateral ureteral obstruction (UUO) in renal tissue and renal interstitial fibroblasts as the research object, to explore the EPO renal tubular interstitial fibrosis mechanism. The results showed that injection of EPO can significantly reduce the renal tubular basement membrane integrity damage, increased expression in obstructive kidneys increased tPA and MMP-9 activity was significantly reduced. After injection of EPO, the content of serum miR-144 was significantly higher in vesicles, microencapsulated EPO mouse serum can inhibit cultured renal interstitial foam matterfrom expression and cell fiber cell tPA activity of MMP-9 in culture fluid, and significantly reduce TGF- beta 1 induced renal interstitial tPA expression and cell culture of microencapsulated MMP-9 activation in the serum of.EPO in liquid foam can fiber cell Reduce the renal tubular basement membrane integrity damage, alleviate the increased expression in obstructive kidneys increased tPA and MMP-9 activity. The negative regulation of renal interstitial fibroblasts and expression of MMP-9 in tPA activity in regulating the expression of miR-144. Overexpression of miR-144 can antagonize the inhibition of miR-144, and aggravated obstruction TGF- beta 1 induced renal interstitial tPA expression and activation of MMP-9 cells cultured in fiber cells. This study suggests that EPO content through upregulation of miR-144 vesicles in serum, inhibit the obstruction of renal tissue into the expression and activation of MMP-9 cells in tPA, reduce the integrity of the tubular basement membrane damage, thus delaying the renal tubule interstitial fibrosis.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R692

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2 王娜娜;microRNA進(jìn)化關(guān)系及編碼特性研究[D];內(nèi)蒙古大學(xué);2007年

3 王玫;小麥microRNA的鑒定與分析[D];南昌大學(xué);2007年

4 秦保東;原發(fā)性膽汁性肝硬化microRNA表達(dá)譜的檢測(cè)及其功能研究[D];第二軍醫(yī)大學(xué);2013年

5 吳曉妍;基于納米復(fù)合材料及生物放大技術(shù)構(gòu)建的電化學(xué)microRNA傳感器的研究[D];西南大學(xué);2015年

6 周景輝;2型糖尿病microRNA表達(dá)譜及其信號(hào)通路研究[D];華南理工大學(xué);2015年

7 姜溪;血漿microRNA-486、microRNA-499在肺癌中的表達(dá)及臨床價(jià)值的研究[D];河北大學(xué);2015年

8 林杉;營(yíng)養(yǎng)誘導(dǎo)舍飼綿羊非繁殖季節(jié)發(fā)情相關(guān)microRNA篩選及其靶基因功能驗(yàn)證[D];石河子大學(xué);2015年

9 林妹妹;microRNA-192 和 microRNA-21 在 IBD 患者中的表達(dá)情況[D];福建醫(yī)科大學(xué);2015年

10 徐嬌陽(yáng);循環(huán)microRNA用于低氧性肺動(dòng)脈高壓早期診斷的實(shí)驗(yàn)研究[D];石河子大學(xué);2015年

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