包含microRNA的微囊泡介导肾小管间质纤维化的机制研究

发布时间：2018-01-29 02:48

本文关键词： microRNA 微囊泡肾小管间质纤维化肾小管上皮细胞表型转化 PTEN microRNA 微囊泡成纤维细胞凋亡肾小管间质纤维化 Bcl-2 促红细胞生成素 microRNA 微囊泡肾小管基底膜组织纤溶酶原激活剂　出处：《南京医科大学》2014年博士论文　论文类型：学位论文

【摘要】：无论原发病因如何,大多数慢性肾脏病最终将进展至肾小管间质纤维化。临床上,肾小管间质纤维化的病变通常呈现由局部到弥漫和不可逆的特征,且病程的进展与原发病因无关。在梗阻性肾小管间质纤维化的动物模型中,肾小管上皮细胞的表型转化和肾小管间质纤维化的病变也表现为局灶起病和弥漫进展,且病变加重的速率与梗阻压力的增速无关。肾小管上皮细胞在肾小管间质纤维化中发挥重要作用,本研究探讨损伤的肾小管上皮细胞本身是否促进了肾小管间质纤维化的进展。以单侧输尿管梗阻(UUO)建立肾小管间质纤维化动物模型和培养的近端肾小管上皮细胞(NRK-52E)系为研究对象,发现损伤的肾小管上皮细胞分泌的微囊泡(MV)能够诱导肾小管上皮细胞发生表型转化；肾小管上皮细胞损伤后,miR-21的表达显著增加并被分泌到微囊泡中；微囊泡将具备生物学活性的外源性miR-21传递到肾小管上皮细胞后,通过抑制靶蛋白PTEN而增强Akt信号诱导肾小管上皮细胞表型转化。本研究从微囊泡介导的细胞间miRNA传递的角度探讨肾小管间质纤维化进展的机制,将有助于研发新的慢性肾脏病的治疗靶点。肾纤维化以肾小管萎缩和细胞外基质蓄积为特征,小管上皮细胞凋亡是肾小管萎缩和间质纤维化的病因之一,然而,肾脏小管上皮细胞凋亡的调节机制仍不清楚。MicroRNA是一类内源性的非编码小RNA,调节细胞的增殖分化代谢和死亡等过程,研究表明microRNA(miR)-34a调节细胞凋亡。MicroRNA不仅能够调节自身细胞的靶基因表达,还通过微囊泡传递来调节其他细胞的靶基因表达。本研究以梗阻性肾纤维化模型及培养的肾小管上皮细胞和肾间质成纤维细胞为研究对象,探讨包含miR-34a的微囊泡调控肾小管上皮细胞凋亡的机制。发现在梗阻的肾组织中,miR-34a的表达增高并主要分布在肾小管间质细胞中。肾组织的微囊泡中miR-34a的表达在梗阻后显著增高。TGF-β1处理上调了肾间质成纤维细胞而非近端肾小管上皮中miR-34a的表达。肾间质成纤维细胞分泌包含miR-34a的微囊泡,经破损的小管基底膜传递到近端肾小管上皮细胞中,通过抑制Bcl-2的表达诱导细胞凋亡。本研究从包含microRNA的微囊泡介导细胞间通讯的角度,为认识肾小管上皮细胞的凋亡提供新的分子机制,为解释肾纤维化的发病机制提供新的理论依据。肾间质纤维化是慢性肾脏病进展的显著特征,肾小管基底膜完整性的破坏在肾脏纤维化过程中发挥关键作用。组织纤溶酶原激活剂(tissue plasminogen activator, tPA)通过激活基质金属蛋白酶(matrix metalloproleinases, MMP)-9,介导肾小管基底膜的降解,是调节基底膜完整性的重要因素。tPA是miR-144的靶基因。注射促红细胞生成素(erythropoietin, EPO)后,血清miR-144的水平显著上升,血清中的miRNA可能包含在微囊泡中。有研究发现,EPO能保护肾脏,延缓肾脏纤维化。本研究以单侧输尿管梗阻(UUO)的肾组织和肾间质成纤维细胞为研究对象,探讨EPO延缓肾小管间质纤维化的机制。结果显示,注射EPO能显著减轻梗阻肾的小管基底膜完整性的破坏,梗阻肾组织中tPA的表达增高和MMP-9的活性增加得到显著减轻。小鼠注射EPO后,血清微囊泡中miR-144的含量显著增高,EPO小鼠血清的微囊泡能抑制培养的肾间质成纤维细胞中tPA的表达和细胞培养液中MMP-9的活性,并显著减轻TGF-β1诱导的肾间质成纤维细胞中tPA表达和细胞培养液中MMP-9活化。EPO小鼠血清的微囊泡能减轻梗阻肾的小管基底膜完整性的破坏,缓解梗阻肾组织中tPA的表达增高和MMP-9的活性增加。调节miR-144的表达能负性调控肾间质成纤维细胞中tPA的表达和MMP-9的活性。过表达miR-144能拮抗,而抑制miR-144则加重梗阻TGF-β1诱导的肾间质成纤维细胞中tPA表达和细胞培养液中MMP-9活化。本研究提示EPO通过上调血清微囊泡中miR-144的含量,抑制梗阻肾组织的成纤维细胞中tPA的表达和MMP-9的活化,减轻小管基底膜完整性的破坏,从而延缓肾小管间质纤维化的进展。
[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.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

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