Niban在肾间质纤维化中的研究

发布时间：2018-06-11 23:17

本文选题：肾间质纤维化 + 蛋白质组学　；参考：《中南大学》2014年博士论文

【摘要】：第一章肾间质纤维化差异蛋白的筛选研究背景：肾间质纤维化(Renal Interstitial Fibrosis, RIF)以肾小管扩张和间质纤维化为特征。研究认为,RIF的程度预示着肾功能受损的程度,与病人肾脏疾病的预后密切相关。RIF是各种慢性肾脏病(Chronic Kidney Disease, CKD)向终末期进展过程中的一种共同病理表现。研究RIF的作用机制,寻找RIF的生物标志物,探索防治RIF的有效措施,对于延缓慢性肾脏疾病的进程意义重大,是目前全球肾病研究的热点和难点。肾间质纤维化的发生、发展受多方面因素的影响,肾小管上皮细胞凋亡、炎症反应、氧化应激、成纤维细胞增殖与活化、肾小管上皮细胞转分化、细胞因子及血管活性物质的产生、细胞外基质合成和降解失衡等均参与其过程。鉴于肾间质纤维化机制的复杂性,我们认为目前已有的研究结果并不能完全解释肾间质纤维化的发病过程,应该还存在尚未引起重视的、可以影响肾间质纤维化过程的新蛋白、新机制。本实验拟通过联合蛋白质组学及基因芯片研究的方法,对肾间质纤维化经典模型——单侧输尿管梗阻(Unilateral Ureteral Obstruction, UUO)大鼠肾组织中的差异蛋白进行筛选,寻找肾间质纤维化过程的生物标志物。目的：在3天、7天、14天、21天UUO大鼠肾组织中,以蛋白质组学联合基因芯片的分析方法,筛选肾间质纤维化过程中有意义的差异蛋白。方法：建立肾间质纤维化UUO大鼠模型。SD大鼠(25只)随机分为假手术组、3天、7天、14天、21天UUO模型组(每组5只)；于UUO术后3、7、14、21天留取各组大鼠梗阻侧肾脏标本,行HE染色观察大鼠肾间质纤维化程度。MASSON染色、III型胶原免疫组化横断面观察14天UUO大鼠肾间质纤维化成模情况。同位素标记相对和绝对定量(Isobaric Tags for Relative and Absolute Quantitation, iTRAQ)技术筛选3天、7天、14天、21天UUO大鼠肾组织差异蛋白表达谱,并与基因芯片的结果进行配对分析,寻找肾间质纤维化过程中有意义的差异蛋白。结果：(1)HE染色结果提示UUO模型成功,3天、7天、14天、21天UUO大鼠肾组织按时间呈现不同程度的肾小管扩张及间质纤维化。14天UUO组大鼠肾脏的肾间质损伤指数、细胞外基质以及Ⅲ型胶原表达量均比假手术组明显升高(p0.05)。 (2)鉴于我们主要着眼于寻找肾间质纤维化早中期的生物标志物,我们以iTRAQ联合基因芯片筛选3天、7天、14天UUO大鼠肾组织中有意义的差异蛋白。与假手术组比较,UUO术后3天、7天、14天四个时间点,差异蛋白持续上/下调(蛋白表达改变1.2or0.8),且满足相对应的基因表达2.0or0.5的蛋白,共15个；其中持续上调的差异蛋白共9个；持续下调的差异蛋白共6个(包括Niban)。 (3) Niban与肾间质纤维化的关系尚不明确,故确定Niban为本研究组后续研究的目标蛋白,重点研究Niban在肾间质纤维化中的表达及可能的作用机制。第二章Niban在肾间质纤维化中的表达研究背景：Niban是2000年由Majima首次发现的蛋白,主要在细胞质中表达。Niban最早在Eker大鼠(一种遗传性肾脏肿瘤的大鼠模型)中发现。在人和动物的其他肾脏肿瘤、患者头颈部鳞癌、头颈部发育不良组织、酗酒／嗜烟及甲状腺损伤患者粘膜上皮组织中,Niban均表达升高。Niban可能作为一种早期肿瘤标志物,对细胞起促进增殖、抑制凋亡的作用。基于本研究组前期实验中Niban在蛋白质组学及基因芯片中的结果,本章节拟对Niban与肾间质纤维化的关系进行验证。目的：通过病人、动物及细胞实验,进一步以体内外实验对肾间质纤维化中Niban蛋白及mRNA表达进行验证。方法：免疫组织化学法对梗阻性肾病患者肾组织中Niban蛋白表达进行定位及半定量检测；免疫组织化学法、Western Blot法对UUO大鼠肾组织中Niban蛋白表达进行定位及半定量检测；Real time-PCR检测Niban mRNA的表达。以TGF-β1(10ng/ml)孵育HK-2细胞48小时,诱导HK-2细胞凋亡,构建体外肾间质纤维化模型。Western Blot法检测TGF-β1诱导的HK-2细胞中Niban、FN的表达,进一步在体外实验中验证肾间质纤维化时Niban的表达情况,FN评估HK-2细胞纤维化状态。结果：1)梗阻性肾病患者肾组织中Niban表达较正常对照组明显减少；2)UUO大鼠肾组织中Niban蛋白表达较假手术组明显减少；而UUO大鼠肾组织中Niban mRNA表达较假手术组升高；3)TGF-β1孵育HK-2细胞48小时后,FN表达升高,Niban表达减少。结论：1)Niban在UUO大鼠及梗阻性肾病患者肾组织中表达减少；2)Niban在TGF-β1诱导的体外肾间质纤维化模型(HK-2)中表达减少。第三章Niban在肾间质纤维化中的作用机制探讨研究背景：肾间质纤维化以肾小管扩张和间质纤维化为特征,与慢性肾脏病的进展密切相关。肾间质纤维化的程度预示着肾功能受损的程度,与患者肾脏疾病的预后关系密切。肾间质纤维化的作用机制复杂,涉及到多个步骤及环节,肾小管上皮细胞凋亡、转分化、巨噬细胞浸润、炎症、氧化应激、多种细胞因子及血管活性物质产生等均参与肾间质纤维化的过程。其中,凋亡在诱发肾小管上皮细胞萎缩及肾间质纤维化形成中起重要作用。凋亡(Apoptosis)也称为“程序性细胞死亡”或“细胞自杀”,是一种遗传控制的生理性死亡。在正常情况下,凋亡起消除老化细胞或未参与免疫反应的淋巴细胞的作用；而发生病理性改变时,凋亡则可以对肿瘤生成在内的多种病理情况起作用。时至今日,已经在多种肾病模型和临床患者肾脏病理检测中,观察到了肾小管上皮细胞的凋亡现象。 Niban的相关研究显示,它是一种功能蛋白。Niban可以通过影响真核转录起始因子——eIF2α及S6K1／4E.BP1磷酸化,在转录水平调节细胞死亡信号；Niban还通过竞争性拮抗MDM2(AKT通路下游的一个作用底物),导致P53降解增多,发挥抑制凋亡的作用.Niban在肾纤维化领域的作用研究,尚不明确。基于Niban对肿瘤细胞凋亡的调控作用,以及上一章我们对Niban在肾间质纤维化中表达的验证,本章节拟对Niban在肾小管上皮细胞凋亡中的作用进行观察,以探讨Niban在肾间质纤维化中的可能作用机制。目的：观察UUO大鼠肾组织及TGF-β1(10ng/ml)孵育48小时后肾小管上皮细胞(HK-2)凋亡情况；观察娲iban表达量改变(沉默或过表达)时,对肾小管上皮细胞凋亡的影响。方法：TUNEL法检测UUO大鼠肾小管上皮细胞凋亡情况；TUNEL法及流式细胞仪检测TGF-β1孵育48小时后HK-2细胞凋亡情况；TUNEL法及流式细胞仪检测siRNA沉默Niban后HK-2细胞凋亡情况；Western Blot法对siRNA沉默Niban的效果进行检测；流式细胞仪检测Niban质粒转染后HK-2细胞凋亡情况。结果：1)与假手术组相比,UUO组大鼠肾小管上皮细胞凋亡明显增加；2)与对照组相比,TGF-β1孵育后HK-2细胞凋亡明显增多；3) siRNA沉默Niban后,HK-2细胞凋亡增加,与对照组相比,差异有统计学意义；4)Niban质粒转入HK-2细胞,TGF-β1干预后细胞凋亡未见减少。结论：1)肾间质纤维化时,肾小管上皮细胞凋亡增加,Niban表达减少,两者趋势相反；2)沉默Niban的表达,可以引起HK-2细胞凋亡增加,提示Niban可能参与肾小管上皮细胞凋亡所致的肾间质纤维化；3)Niban在肾间质纤维化中对HK-2细胞凋亡的调节,不通过TGF-β1通路。
[Abstract]:The first chapter of screening differential proteins of renal interstitial fibrosis
Background: Renal Interstitial Fibrosis (RIF) is characterized by tubulodilatation and interstitial fibrosis. The study suggests that the degree of RIF is an indication of the extent of renal impairment and is closely related to the prognosis of renal diseases in patients with the progression of various chronic renal diseases (Chronic Kidney Disease, CKD) to the terminal stage. A common pathological manifestation. The study of the mechanism of the action of RIF, the search for the biomarkers of RIF and the exploration of the effective measures for the prevention and control of RIF are of great significance for delaying the process of chronic renal disease. It is a hot and difficult point in the study of kidney disease in the world at present. The development of renal interstitial fibrosis is affected by many factors, and the apoptosis of renal tubular epithelial cells is the same. Inflammatory reaction, oxidative stress, fibroblast proliferation and activation, renal tubular epithelial cells transdifferentiation, cytokine and vasoactive substances production, extracellular matrix synthesis and degradation imbalance are involved in the process. In view of the complexity of the mechanism of renal interstitial fibrosis, we believe that the present results do not fully explain the renal interstitium. There should be a new protein and new mechanism that can affect the process of renal interstitial fibrosis. This experiment is intended to be a classic model of renal interstitial fibrosis (Unilateral Ureteral Obstruction, UUO) by combining proteomics and gene chip research. The differentially expressed proteins in renal tissue were screened for biomarkers in renal interstitial fibrosis.
Objective: in the renal tissue of UUO rats, 3 days, 7 days, 14 days and 21 days, the significance of differential protein in the process of renal interstitial fibrosis was screened by the analysis of proteomics and gene chip.
Methods: the UUO rat model of renal interstitial fibrosis (25 rats) was randomly divided into sham operation group, 3 days, 7 days, 14 days, and 21 days UUO model group (5 rats in each group). After 3,7,14,21 days after UUO, the rats were left with the obstructive side kidney specimens. HE staining was used to observe the degree of renal interstitial fibrosis,.MASSON staining, and III type collagen immunohistochemical cross-sectional view. Detection of renal interstitial fibrosis in 14 days of UUO rats. The relative and absolute quantitative markers (Isobaric Tags for Relative and Absolute Quantitation, iTRAQ) were used to screen the differential protein expression profiles of renal tissue in UUO rats for 3 days, 7 days, 14 days and 21 days, and paired analysis with the results of gene chip to find the process of renal interstitial fibrosis. Meaningful differential protein.
Results: (1) HE staining results showed that UUO model was successful, 3 days, 7 days, 14 days, and 21 days of UUO rats showed renal interstitial injury index of renal tubules in different degrees of renal tubule dilatation and interstitial fibrosis on.14 days of UUO group, and the expression of extracellular matrix and type III collagen was significantly higher than that of sham operation group (P0.05).
(2) in view of our main focus on finding biomarkers in the early and middle stages of renal interstitial fibrosis, we screened the significant difference proteins in the renal tissue of UUO rats by iTRAQ combined gene chip for 3 days, 7 days and 14 days. Compared with the sham operation group, the difference in protein expression was maintained at 3 days, 7 days and 14 days at four time points after UUO, and the difference in protein expression was on / down (1.2or0.8). A total of 15 proteins that meet the corresponding gene expression 2.0or0.5, 9 of which are continuously up-regulated, 6 (including Niban).
(3) the relationship between Niban and renal interstitial fibrosis is not clear, so Niban is the target protein for follow-up study in this study group, and the expression of Niban in renal interstitial fibrosis and the possible mechanism of action are focused on.
The expression of Niban in renal interstitial fibrosis in the second chapter
Background: Niban was the first protein found by Majima in 2000. The expression of.Niban in cytoplasm was first found in Eker rats (a rat model of a hereditary renal tumor). In human and animal other renal tumors, patients with head and neck squamous cell carcinoma, undeveloped head and neck tissues, alcoholism / smoke and thyroid injury patients' mucous membrane were found. In epithelial tissue, Niban expressed that elevated.Niban may be an early tumor marker, which promotes proliferation and inhibits apoptosis. Based on the results of Niban in proteomics and gene chips in earlier experiments of this study group, this chapter is to verify the relationship between Niban and renal interstitial fibrosis.
Objective: to verify the expression of Niban protein and mRNA in renal interstitial fibrosis by in vivo and in vitro experiments through patient, animal and cell experiments.
Methods: immunohistochemical method was used to locate and semi quantitative detection of Niban protein expression in renal tissue of patients with obstructive nephropathy. Immunohistochemical method and Western Blot method were used to locate and semi quantitative detection of Niban protein expression in renal tissue of UUO rats; Real time-PCR was used to detect the expression of Niban mRNA. TGF- beta 1 (10ng/ml) was used to incubate HK-2 fine. To induce apoptosis of HK-2 cells for 48 hours, the expression of Niban and FN in HK-2 cells induced by TGF- beta 1 was detected by.Western Blot method in vitro, and the expression of Niban in renal interstitial fibrosis was verified in vitro, and FN evaluated the fibrosis state of HK-2 cells in FN.
Results: 1) the expression of Niban in the renal tissue of patients with obstructive nephropathy was significantly lower than that in the normal control group; 2) the expression of Niban protein in the renal tissue of UUO rats was significantly lower than that in the sham group, while the expression of Niban mRNA in the renal tissue of UUO rats was higher than that in the sham group; 3) TGF- beta 1 was incubated with HK-2 cells for 48 hours, and the expression of FN increased and the expression of Niban decreased.
Conclusion: 1) the expression of Niban decreased in the renal tissue of UUO rats and patients with obstructive nephropathy, and 2) the expression of Niban decreased in the TGF- beta 1 induced renal interstitial fibrosis model (HK-2).
The third chapter is about the mechanism of Niban in renal interstitial fibrosis.
Background: renal interstitial fibrosis is characterized by tubulodilatation and interstitial fibrosis, which is closely related to the progress of chronic renal disease. The degree of renal interstitial fibrosis indicates the extent of renal impairment and is closely related to the prognosis of renal diseases. The mechanism of renal interstitial fibrosis is complex, involving multiple steps and links, and kidney. Apoptosis, transdifferentiation, macrophage infiltration, inflammation, oxidative stress, and the production of various cytokines and vasoactive substances are involved in the process of renal interstitial fibrosis, among which apoptosis plays an important role in inducing renal tubular epithelial cell atrophy and renal interstitial fibrosis.
Apoptosis (Apoptosis), also known as "programmed cell death" or "cell suicide", is a genetic control of physiological death. In normal cases, apoptosis can eliminate the effects of aging cells or lymphocytes that do not participate in the immune response; and when pathological changes occur, death can be found in a variety of pathological conditions. To date, the apoptosis of renal tubular epithelial cells has been observed in a variety of Nephropathy Models and clinical patients.
Niban related studies have shown that it is a functional protein.Niban that can modulate cell death signals at transcriptional levels by affecting the eukaryotic transcription initiation factor, eIF2 alpha and S6K1 / 4E.BP1 phosphorylation, and Niban also leads to an increase in P53 degradation by competitive antagonism of MDM2 (a substrate downstream of AKT pathway) and the inhibition of apoptosis. The role of.Niban in the field of renal fibrosis is not yet clear. Based on the role of Niban in the regulation of apoptosis of tumor cells, and the previous chapter we verify the expression of Niban in renal interstitial fibrosis, this chapter is to observe the role of Niban in renal tubular epithelial cell apoptosis in order to explore the possibility of Niban in renal interstitial fibrosis. Mechanism of action.
Objective: To observe the apoptosis of renal tubular epithelial cells (HK-2) after incubation of renal tissue and TGF- beta 1 (10ng/ml) in UUO rats for 48 hours, and to observe the effect of the change of the expression of Wa Iban (silence or overexpression) on the apoptosis of renal tubular epithelial cells.
Methods: TUNEL assay was used to detect the apoptosis of renal tubular epithelial cells in UUO rats; TUNEL and flow cytometry were used to detect the apoptosis of HK-2 cells after TGF- beta 1 was incubated for 48 hours; TUNEL method and flow cytometry were used to detect the apoptosis of HK-2 cells after siRNA silencing Niban; Western Blot method was used to detect the effect of siRNA silence; flow cytometry The apoptosis of HK-2 cells after transfection of Niban plasmid was detected.
Results: 1) compared with the sham operation group, the apoptosis of renal tubular epithelial cells in the UUO group was significantly increased; 2) the apoptosis of HK-2 cells increased significantly after the incubation of TGF- beta 1 compared with the control group; 3) after siRNA silencing Niban, the apoptosis of HK-2 cells increased, and the difference was statistically significant compared with the control group; 4) Niban plasmid was transferred to HK-2 cells and TGF- beta 1 had the prognosis of cells. No decrease in apoptosis was found.
Conclusions: 1) during renal interstitial fibrosis, the apoptosis of renal tubular epithelial cells increased and the expression of Niban decreased. 2) the expression of silent Niban could induce the increase of apoptosis in HK-2 cells, suggesting that Niban may be involved in renal interstitial fibrosis due to apoptosis of renal tubular epithelial cells; 3) Niban apoptosis of HK-2 cells in renal interstitial fibrosis. Regulation, not through TGF- beta 1 pathway.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

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