【作者】 熊俊；

【导师】 刘厚奇；

【作者基本信息】 第二军医大学 ， 人体解剖与组织胚胎学， 2011， 博士

【摘要】 慢性肾脏疾病(Chronic Kidney Disease,CKD)常因起病隐匿,面临着高发病率、高住院率、高死亡率及高额治疗费用等问题,已成为威胁人类健康的隐形杀手,对人类生存质量及社会发展构成极大危害。目前认为,CKD的发展是一种不可逆的过程,最终将形成广泛的肾间质纤维化,并发展为终末期肾衰。而肾间质纤维化(Renal Interstitial Fibrosis)被认为是所有慢性进行性肾脏疾病进展到终末期肾衰的共同组织学特点,在CKD进程中起主导作用。肾间质纤维化特征性的表现为细胞外基质(Extracellular Matrix,ECM)聚集和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)阳性肌成纤维细胞(Myofibroblast)的大量增殖。而肌成纤维细胞被认为是肾间质纤维化过程中ECM合成和沉积的关键效应细胞,在CKD中起重要作用,但其确切来源目前仍不清楚。1995年,Strutz等首次提出肾间质纤维化过程中存在上皮-间充质转型(Epithelial-Mesenchymal Transition,EMT)现象,并认为肾间质纤维化过程中出现的肌成纤维细胞是由肾小管上皮细胞发生EMT产生的,但此观点至今仍无定论。为此,本课题也试图通过体外诱导人近端肾小管上皮细胞HK-2细胞和体内小鼠的单侧输尿管梗阻(Unilateral Ureteral Obstruction, UUO)模型来研究肾间质纤维化过程中是否存在EMT现象及其分子机制。在体外细胞模型研究中,本课题首先从形态学上观察了TGF-β1(5ng/ml)、EGF(10ng/ml)单独或TGF-β1(5ng/ml)/EGF(10ng/ml)联合作用对HK-2细胞的影响。结果显示,TGF-β1单独或TGF-β1/EGF联合诱导HK-2细胞后,HK-2细胞在形态上均发生了EMT现象,而且TGF-β1/EGF联合诱导的效果更理想。据此,本研究选择了TGF-β1/EGF联合诱导HK-2细胞作为体外研究肾小管上皮细胞发生EMT现象的模型。细胞划痕实验和细胞迁移实验发现,TGF-β1/EGF联合作用HK-2细胞后,细胞的运动速度和迁移速度均明显增加,提示TGF-β1/EGF联合作用HK-2细胞后,细胞的表型可能发生了EMT转变。进一步采用免疫荧光细胞化学法检测TGF-β1/EGF联合诱导HK-2细胞后,上皮细胞标志CK18、E-cadherin和间质细胞标志Vimentin的表达情况。结果发现,诱导后的HK-2细胞上皮细胞分子标志CK18和E-cadherin均明显下降,而间质细胞分子标志Vimentin的表达则升高,从上皮标志物下调和间充质标志物上调判断TGF-β1/EGF联合诱导HK-2细胞确实发生了EMT。为了探讨TGF-β1/EGF联合诱导HK-2细胞发生EMT的分子机制,本研究通过RT-PCR和免疫荧光细胞化学法检测了EMT相关的分子。结果发现,转录因子Snail1的转录和表达水平在TGF-β1/EGF联合诱导HK-2细胞发生EMT的过程中明显升高,提示Snail1参与了TGF-β1/EGF诱导HK-2细胞发生EMT的过程。进一步通过免疫印迹研究发现,TGF-β1/EGF诱导后的HK-2细胞中,ERK1(p44)/ERK2(p42)的磷酸化水平很高,提示TGF-β1/EGF联合诱导的HK-2细胞中Snail1的高表达是通过ERK通路调控的,即ERK通路介导调控Snail1的增高,从而导致了TGF-β1/EGF联合诱导HK-2细胞发生明显EMT。为了进一步证实ERK通路介导了TGF-β1/EGF联合诱导的HK-2细胞中Snail1的高表达,本研究利用ERK通路抑制剂U0126,研究其对TGF-β1/EGF联合诱导后的HK-2细胞中,Snail1转录和表达水平以及细胞形态和运动的影响。结果显示,ERK通路抑制剂U0126抑制了TGF-β1/EGF诱导后HK-2细胞中ERK1/2的磷酸化水平以及Snail1的蛋白表达水平,同时抑制了TGF-β1/EGF诱导的HK-2细胞发生的形态和运动改变,但并未抑制Snail1的转录水平,提示U0126可通过抑制ERK通路中p44/42的磷酸化水平,进而在蛋白水平抑制Snail1的表达量,最终抑制了TGF-β1/EGF诱导的HK-2细胞发生EMT的过程。在体内小鼠肾间质纤维化模型研究中,本课题首先建立了小鼠的UUO模型,在此基础上本研究着重对小鼠UUO术后14天前的手术侧肾脏进行了组织病理学研究,结果显示,小鼠UUO术后,手术侧肾脏随术后时间的增加,肾间质纤维化的程度越来越严重,提示小鼠UUO模型可用于小鼠肾间质纤维化的研究。进一步通过免疫荧光组织化学和免疫印迹等方法研究了体内肾间质纤维化过程中是否存在EMT及EMT相关分子的变化情况,结果显示,UUO术后肾间质纤维化过程中,上皮细胞标志物E-cadherin的表达降低,肌成纤维细胞的标志物α-SMA增加,同时观察到少量肾小管上皮细胞表达α-SMA,表明小鼠体内肾间质纤维化过程中存在EMT现象。对肾间质纤维化过程中EMT相关分子的检测发现,明胶酶MMP2和MMP9在14天内有增高,提示明胶酶可能参与纤维的降解和平衡,并有助于促进肾小管细胞通过基底膜发生迁移;肾小管上皮细胞中表达PAX2阳性细胞增多,提示成熟的肾小管上皮细胞在肾间质纤维化进程中,逐渐转变为后肾间充质样细胞;研究还发现,在肾间质纤维化的过程中皮质间质内出现了表达BMP7的细胞,其细胞类型和作用有待进一步证实;转录因子Snail1持续上调,提示Snail1在体内肾间质纤维化中具有重要作用。为了更好的研究转录因子Snail1瞬时高表达对HK-2细胞的表型影响,本研究还构建了人Snail1的重组腺病毒载体,并成功的包装了腺病毒,Snail1重组腺病毒感染HK-2细胞后,形态观察显示,细胞的形态由上皮样转变为成纤维样。通过RT-PCR、免疫印迹和免疫荧光细胞化学的方法证实,Snail1重组腺病毒感染的细胞中,上皮标志E-cadherin的表达减少,与E-cadherin相关的β-catenin从细胞膜转位到细胞质,肌成纤维细胞标志α-SMA上调,明胶酶MMP2和MMP9的转录水平较高。提示所获得的腺病毒可以直接导致HK-2细胞发生EMT现象,这为后续将进行的Snail1对下游分子的调控研究奠定了基础。总之,本研究通过体内外的手段研究了肾间质纤维化过程中的EMT现象并探讨了Snail1在其中的作用,研究结果得出以下结论:①TGF-β1/EGF联合诱导可导致人肾近端小管上皮细胞HK-2细胞发生明显的EMT现象,其分子机制是TGF-β1/EGF协同作用进一步促进ERK1/2(p44/42)的磷酸化,ERK通路进一步上调Snail1,从而导致肾小管上皮细胞发生明显EMT现象;抑制ERK1/2(p44/42)的磷酸化,可以抑制Snail1的蛋白水平,从而抑制EMT。这具有一定的创新性,进一步丰富了人们对肾小管上皮细胞发生EMT的分子机制的认识。②小鼠体内肾间质纤维化过程中也存在EMT现象,其分子机制涉及明胶酶MMP2和MMP9、PAX2、BMP7和Snail1等多个分子,并首次发现在肾间质纤维化中存在表达BMP7的细胞,其细胞类型和作用有待进一步证实。③Snail1瞬时过表达导致了肾小管上皮细胞HK-2细胞直接发生EMT,这将为人们寻找延缓或阻止肾间质纤维化提供新思路和新靶点。本研究的创新之处在于:①本研究首次发现,TGF-β1/EGF联合诱导人肾近端小管上皮细胞HK-2细胞发生EMT的分子机制是:TGF-β1/EGF协同作用促进ERK1/2(p44/42)的磷酸化,ERK通路进一步上调Snail1,从而导致肾小管上皮细胞发生明显EMT现象。②在研究小鼠体内肾间质纤维化过程中的EMT现象时,本研究首次发现在肾间质纤维化中存在表达BMP7的细胞,其细胞类型和作用有待进一步证实。更多还原

【Abstract】 Chronic kidney disease (CDS) is a worldwide threat to public health, with increasing prevalence, high costs, and poor prognosis. Progressive tubulointerstitial fibrosis is the terminal pathway for all CDS leading to chronic renal failure (CRF). There have been many studies stressing that tubulointerstitial fibrosis, characterized by loss of renal tubules and interstitial capillaries and the accumulation of extracellular matrix proteins, is responsible for renal dysfunction rather than glomerular changes. Fibroblasts and myofibroblasts are considered to be the key effector cells in renal tubulointerstitial fibrosis responsible for the synthesis and deposition of extracellular matrix components. In 1990, Gown claimed to have the mysteries of the myofibroblast (partially at least) unmasked. But 20 years later, the origin of activated ?broblasts and myo?broblasts still remains controversial. There are several potential sources for myofibroblasts in renal tubulointerstitial fibrosis than tissue-resident fibroblasts, involving circulating cells of bone marrow origin (fibrocytes), vascular pericytes, endothelial cells, and tubular epithelial cells. Since 1995, the transition of tubular epithelial cells to a mesenchymal phenotype (epithelial-mesenchymal transition, EMT) in the renal cortex has been firstly reported by Strutz et al, however, 15 years later, researchers are still continue to study the phenomenon and mechanism of EMT. In response to injury and inflammatory stimuli, it is proposed that epithelial cells undergo morphological changes, losing epithelial characteristics such as polarity and the expression of junctional markers, while inducing fibroblast markers such as FSP-1 (fibroblast specific protein 1), vimentin andα-SMA (α-smooth muscle actin). The cells begin to express stress fibres containingα-SMA and migrate through the basement membrane to become myofibroblasts in the interstitium. However, it is still controversial whether myofibroblasts were derived from tubular epithelial cells via EMT in renal tubulointerstitial fibrosis. In our study, we also tried to observe the phenomenon of EMT in vivo using mice unilateral ureteral obstruction (UUO) model and in vitro culturing immortalized human proximal tubular epithelial HK-2 cells.In in vitro study, we first observed the morphological change of HK-2 cells induced by TGF-β1 (5ng/ml) or EGF (10ng/ml) alone, and the combinating administration of TGF-β1 (5ng/ml) and EGF (10ng/ml). It is showed that the combinating use of EGF and TGF-β1 could more effectively stimulate the morphologic alteration of HK-2 cells from epithelial to mesenchymal phenotype when compared to TGF-β1 or EGF alone. Further, a follow-up study chose HK-2 cells induced by the combinating use of EGF and TGF-β1 as a model of renal tubular epithelial cells EMT in vitro. Using scratch test and matrigel invasion assays, we found that the cell migration velocity of HK-2 cells significantly increased when induced by the combinating use of EGF and TGF-β1. We also observed that the epithelial cell markers CK18 and E-cadherin was downregulated while mesenchymal cells marker Vimentin was upregulated using immunofluorescence. In conclusion, HK-2 cells have undertaken EMT when induced by the combinating use of EGF and TGF-β1.In order to explore the molecular mechanism of EMT, we then detected the EMT-related molecules’expressions via RT-PCR and immunofluorescence. We found that the transcriptive and expressing level of transcription factor Snail1 increased in HK-2 cells during EMT induced by the combinating use of EGF and TGF-β1. Western blot analysis also confirmed that ERK (p44, ERK1 and p42, ERK2) phosphorylation were more active and Snail1 expression level were higher when compared HK-2 cells that undertook EMT induced by the combinating use of EGF and TGF-β1 to those administrated with TGF-β1 or EGF alone. All the above suggested that ERK pathway mediates the increase of transcription factor Snail1 which may contribute to HK-2 cells EMT process.To further confirm the effect of ERK Pathway on Snail1 expression, MEK inhibitor U0126 was used to examine its impact on HK-2 cells EMT induced by the combinating use of EGF and TGF-β1. The results showed that MEK inhibitor U0126 could effectively block ERK1/2 phosphorylation, inhibited the expression of Snail1 while simutaneously inhibited the morphologic alteration and movement of HK-2 cells induced by the combinating use of EGF and TGF-β1. However, the level of Snail1 transcription is not inhibited in HK-2 cells using MEK inhibitor U0126. It is suggested that U0126 blocked ERK pathway as well as Snail1 protein expression, and subsequently inhibit TGF-β1/EGF-induced HK-2 cells EMT process.For in vivo studies, we used mice UUO-induced renal interstitial fibrosis model to observe the pathological changes of kidneys within 14 days after UUO establishment. The model mice suffered from renal interstitial fibrosis were identified by renal histological examinations. By further demonstration given by immunofluorescence and Western blottings, we observed that epithelial cell markers E-cadherin expression decrease in contrast to myofibroblast markerα-SMA increase. The increase ofα-SMA-positive cells were mainly located around small blood vessels at early stage on Day 4 after UUO establishment. On Day 14, we observed a few renal tubular epithelial cells begin to expressα-SMA, which demonstrated that EMT phenomenon indeed exist during renal interstitial fibrosis in mice. Gelatin zymographic analysis showed the expression of MMP2 and MMP9 were increased temporarily in early stage of renal interstitial fibrosis, suggesting that gelatinase may be involved in the degradation and balance of fibers in renal interstitium during this period, and help promoting renal tubular cells migration through the basement to interstitium. The number of PAX2-positive cells was increased among renal tubular epithelial cells detected by immunofluorescence, suggesting that renal tubular epithelial cells may first transited to metanephric mesenchymal-like cells. Also by immunofluorescence, we found a amount of BMP7-positive cells began to appear in renal cortical interstitium, we attrbuted this observation to the possible infiltration of mononuclear cells and thus may as a result take renal protective effects, which undoubtedly require further experiments to confirm. The transcriptive and protein level of EMT-related transcription factor Snail1 gradually increased as the time prolonging during this period, indicating that the proportion of the expression of Snail1 cells gradually increased.In additon, we also constructed a adenovirus vector containing human Snail1 and successful packaging of the adenovirus to study the phenotype impact of transient expression of transcription factors Snail1 in HK-2 cells. It was observed that phenotype changed in HK-2 cells infected with adenoviruses, showing a fibroblast-like shape. By RT-PCR, Western blot and immunofluorescence, we found that, compared with the control adenovirus, the expression of the epithelial cells marker E-cadherin reduced, E-cadherin-associatedβ-catenin translocation from the cell membrane to the cytoplasm, myofibroblasts markerα-SMA increased, and gelatinases (MMP2 and MMP9) transcription level were increased. All these demonstrated HK-2 cells infected with adenoviruses containing human Snail1 could lead to EMT.Through this study, the conclusion we could made were listed as follows①the combinating use of EGF and TGF-β1 can lead human renal proximal tubular epithelial cells line HK-2 cells to undertake EMT. The synergy of TGF-β1/EGF could enhance ERK1 / 2 (p44/42) phosphorylation and then increase Snail1. MEK inhibitor U0126 can block ERK1 / 2 (p44/42 ) phosphorylation and Snail1 expression at the protein level, and subsequently inhibit EMT, suggesting ERK pathway regulate the expression of Snail1 at the protein level in HK-2 cells during EMT.②In mice UUO model, myofibroblasts, which appeared firstly around the vessels, derived from perivascular cells or endothelial cells during early stage of renal interstitial fibrosis. The renal tubular epithelial cells transited into metanephric mesenchymal-like cells firstly, then could expressα-SMA and transited into myofibroblasts on Day 14, prompted myofibroblasts increase by EMT during later stage of renal interstitial fibrosis. At the same time, a large number of cortical interstitial mononuclear cells expressed BMP7, suggesting the potential renal protective effects, which require further experiments to confirm.③Transient expression of transcription factors Snail1 in human renal proximal tubular epithelial cell line HK-2 cells could lead to phenotype changes, cell migration, and EMT.更多还原