本文選題：腎間質(zhì)纖維化 + 蛋白質(zhì)組學(xué)　； 參考：《中南大學(xué)》2014年博士論文

【摘要】：第一章腎間質(zhì)纖維化差異蛋白的篩選 研究背景：腎間質(zhì)纖維化(Renal Interstitial Fibrosis, RIF)以腎小管擴(kuò)張和間質(zhì)纖維化為特征。研究認(rèn)為,RIF的程度預(yù)示著腎功能受損的程度,與病人腎臟疾病的預(yù)后密切相關(guān)。RIF是各種慢性腎臟病(Chronic Kidney Disease, CKD)向終末期進(jìn)展過程中的一種共同病理表現(xiàn)。研究RIF的作用機(jī)制,尋找RIF的生物標(biāo)志物,探索防治RIF的有效措施,對于延緩慢性腎臟疾病的進(jìn)程意義重大,是目前全球腎病研究的熱點(diǎn)和難點(diǎn)。腎間質(zhì)纖維化的發(fā)生、發(fā)展受多方面因素的影響,腎小管上皮細(xì)胞凋亡、炎癥反應(yīng)、氧化應(yīng)激、成纖維細(xì)胞增殖與活化、腎小管上皮細(xì)胞轉(zhuǎn)分化、細(xì)胞因子及血管活性物質(zhì)的產(chǎn)生、細(xì)胞外基質(zhì)合成和降解失衡等均參與其過程。鑒于腎間質(zhì)纖維化機(jī)制的復(fù)雜性,我們認(rèn)為目前已有的研究結(jié)果并不能完全解釋腎間質(zhì)纖維化的發(fā)病過程,應(yīng)該還存在尚未引起重視的、可以影響腎間質(zhì)纖維化過程的新蛋白、新機(jī)制。本實(shí)驗(yàn)擬通過聯(lián)合蛋白質(zhì)組學(xué)及基因芯片研究的方法,對腎間質(zhì)纖維化經(jīng)典模型——單側(cè)輸尿管梗阻(Unilateral Ureteral Obstruction, UUO)大鼠腎組織中的差異蛋白進(jìn)行篩選,尋找腎間質(zhì)纖維化過程的生物標(biāo)志物。 目的：在3天、7天、14天、21天UUO大鼠腎組織中,以蛋白質(zhì)組學(xué)聯(lián)合基因芯片的分析方法,篩選腎間質(zhì)纖維化過程中有意義的差異蛋白。 方法：建立腎間質(zhì)纖維化UUO大鼠模型。SD大鼠(25只)隨機(jī)分為假手術(shù)組、3天、7天、14天、21天UUO模型組(每組5只)；于UUO術(shù)后3、7、14、21天留取各組大鼠梗阻側(cè)腎臟標(biāo)本,行HE染色觀察大鼠腎間質(zhì)纖維化程度。MASSON染色、III型膠原免疫組化橫斷面觀察14天UUO大鼠腎間質(zhì)纖維化成模情況。同位素標(biāo)記相對和絕對定量(Isobaric Tags for Relative and Absolute Quantitation, iTRAQ)技術(shù)篩選3天、7天、14天、21天UUO大鼠腎組織差異蛋白表達(dá)譜,并與基因芯片的結(jié)果進(jìn)行配對分析,尋找腎間質(zhì)纖維化過程中有意義的差異蛋白。 結(jié)果：(1)HE染色結(jié)果提示UUO模型成功,3天、7天、14天、21天UUO大鼠腎組織按時(shí)間呈現(xiàn)不同程度的腎小管擴(kuò)張及間質(zhì)纖維化。14天UUO組大鼠腎臟的腎間質(zhì)損傷指數(shù)、細(xì)胞外基質(zhì)以及Ⅲ型膠原表達(dá)量均比假手術(shù)組明顯升高(p0.05)。 (2)鑒于我們主要著眼于尋找腎間質(zhì)纖維化早中期的生物標(biāo)志物,我們以iTRAQ聯(lián)合基因芯片篩選3天、7天、14天UUO大鼠腎組織中有意義的差異蛋白。與假手術(shù)組比較,UUO術(shù)后3天、7天、14天四個(gè)時(shí)間點(diǎn),差異蛋白持續(xù)上/下調(diào)(蛋白表達(dá)改變1.2or0.8),且滿足相對應(yīng)的基因表達(dá)2.0or0.5的蛋白,共15個(gè)；其中持續(xù)上調(diào)的差異蛋白共9個(gè)；持續(xù)下調(diào)的差異蛋白共6個(gè)(包括Niban)。 (3) Niban與腎間質(zhì)纖維化的關(guān)系尚不明確,故確定Niban為本研究組后續(xù)研究的目標(biāo)蛋白,重點(diǎn)研究Niban在腎間質(zhì)纖維化中的表達(dá)及可能的作用機(jī)制。 第二章Niban在腎間質(zhì)纖維化中的表達(dá) 研究背景：Niban是2000年由Majima首次發(fā)現(xiàn)的蛋白,主要在細(xì)胞質(zhì)中表達(dá)。Niban最早在Eker大鼠(一種遺傳性腎臟腫瘤的大鼠模型)中發(fā)現(xiàn)。在人和動(dòng)物的其他腎臟腫瘤、患者頭頸部鱗癌、頭頸部發(fā)育不良組織、酗酒／嗜煙及甲狀腺損傷患者粘膜上皮組織中,Niban均表達(dá)升高。Niban可能作為一種早期腫瘤標(biāo)志物,對細(xì)胞起促進(jìn)增殖、抑制凋亡的作用�；诒狙芯拷M前期實(shí)驗(yàn)中Niban在蛋白質(zhì)組學(xué)及基因芯片中的結(jié)果,本章節(jié)擬對Niban與腎間質(zhì)纖維化的關(guān)系進(jìn)行驗(yàn)證。 目的：通過病人、動(dòng)物及細(xì)胞實(shí)驗(yàn),進(jìn)一步以體內(nèi)外實(shí)驗(yàn)對腎間質(zhì)纖維化中Niban蛋白及mRNA表達(dá)進(jìn)行驗(yàn)證。 方法：免疫組織化學(xué)法對梗阻性腎病患者腎組織中Niban蛋白表達(dá)進(jìn)行定位及半定量檢測；免疫組織化學(xué)法、Western Blot法對UUO大鼠腎組織中Niban蛋白表達(dá)進(jìn)行定位及半定量檢測；Real time-PCR檢測Niban mRNA的表達(dá)。以TGF-β1(10ng/ml)孵育HK-2細(xì)胞48小時(shí),誘導(dǎo)HK-2細(xì)胞凋亡,構(gòu)建體外腎間質(zhì)纖維化模型。Western Blot法檢測TGF-β1誘導(dǎo)的HK-2細(xì)胞中Niban、FN的表達(dá),進(jìn)一步在體外實(shí)驗(yàn)中驗(yàn)證腎間質(zhì)纖維化時(shí)Niban的表達(dá)情況,FN評估HK-2細(xì)胞纖維化狀態(tài)。 結(jié)果：1)梗阻性腎病患者腎組織中Niban表達(dá)較正常對照組明顯減少；2)UUO大鼠腎組織中Niban蛋白表達(dá)較假手術(shù)組明顯減少；而UUO大鼠腎組織中Niban mRNA表達(dá)較假手術(shù)組升高；3)TGF-β1孵育HK-2細(xì)胞48小時(shí)后,FN表達(dá)升高,Niban表達(dá)減少。 結(jié)論：1)Niban在UUO大鼠及梗阻性腎病患者腎組織中表達(dá)減少；2)Niban在TGF-β1誘導(dǎo)的體外腎間質(zhì)纖維化模型(HK-2)中表達(dá)減少。 第三章Niban在腎間質(zhì)纖維化中的作用機(jī)制探討 研究背景：腎間質(zhì)纖維化以腎小管擴(kuò)張和間質(zhì)纖維化為特征,與慢性腎臟病的進(jìn)展密切相關(guān)。腎間質(zhì)纖維化的程度預(yù)示著腎功能受損的程度,與患者腎臟疾病的預(yù)后關(guān)系密切。腎間質(zhì)纖維化的作用機(jī)制復(fù)雜,涉及到多個(gè)步驟及環(huán)節(jié),腎小管上皮細(xì)胞凋亡、轉(zhuǎn)分化、巨噬細(xì)胞浸潤、炎癥、氧化應(yīng)激、多種細(xì)胞因子及血管活性物質(zhì)產(chǎn)生等均參與腎間質(zhì)纖維化的過程。其中,凋亡在誘發(fā)腎小管上皮細(xì)胞萎縮及腎間質(zhì)纖維化形成中起重要作用。 凋亡(Apoptosis)也稱為“程序性細(xì)胞死亡”或“細(xì)胞自殺”,是一種遺傳控制的生理性死亡。在正常情況下,凋亡起消除老化細(xì)胞或未參與免疫反應(yīng)的淋巴細(xì)胞的作用；而發(fā)生病理性改變時(shí),凋亡則可以對腫瘤生成在內(nèi)的多種病理情況起作用。時(shí)至今日,已經(jīng)在多種腎病模型和臨床患者腎臟病理檢測中,觀察到了腎小管上皮細(xì)胞的凋亡現(xiàn)象。 Niban的相關(guān)研究顯示,它是一種功能蛋白。Niban可以通過影響真核轉(zhuǎn)錄起始因子——eIF2α及S6K1／4E.BP1磷酸化,在轉(zhuǎn)錄水平調(diào)節(jié)細(xì)胞死亡信號(hào)；Niban還通過競爭性拮抗MDM2(AKT通路下游的一個(gè)作用底物),導(dǎo)致P53降解增多,發(fā)揮抑制凋亡的作用.Niban在腎纖維化領(lǐng)域的作用研究,尚不明確。基于Niban對腫瘤細(xì)胞凋亡的調(diào)控作用,以及上一章我們對Niban在腎間質(zhì)纖維化中表達(dá)的驗(yàn)證,本章節(jié)擬對Niban在腎小管上皮細(xì)胞凋亡中的作用進(jìn)行觀察,以探討Niban在腎間質(zhì)纖維化中的可能作用機(jī)制。 目的：觀察UUO大鼠腎組織及TGF-β1(10ng/ml)孵育48小時(shí)后腎小管上皮細(xì)胞(HK-2)凋亡情況；觀察媧iban表達(dá)量改變(沉默或過表達(dá))時(shí),對腎小管上皮細(xì)胞凋亡的影響。 方法：TUNEL法檢測UUO大鼠腎小管上皮細(xì)胞凋亡情況；TUNEL法及流式細(xì)胞儀檢測TGF-β1孵育48小時(shí)后HK-2細(xì)胞凋亡情況；TUNEL法及流式細(xì)胞儀檢測siRNA沉默Niban后HK-2細(xì)胞凋亡情況；Western Blot法對siRNA沉默Niban的效果進(jìn)行檢測；流式細(xì)胞儀檢測Niban質(zhì)粒轉(zhuǎn)染后HK-2細(xì)胞凋亡情況。 結(jié)果：1)與假手術(shù)組相比,UUO組大鼠腎小管上皮細(xì)胞凋亡明顯增加；2)與對照組相比,TGF-β1孵育后HK-2細(xì)胞凋亡明顯增多；3) siRNA沉默Niban后,HK-2細(xì)胞凋亡增加,與對照組相比,差異有統(tǒng)計(jì)學(xué)意義；4)Niban質(zhì)粒轉(zhuǎn)入HK-2細(xì)胞,TGF-β1干預(yù)后細(xì)胞凋亡未見減少。 結(jié)論：1)腎間質(zhì)纖維化時(shí),腎小管上皮細(xì)胞凋亡增加,Niban表達(dá)減少,兩者趨勢相反；2)沉默Niban的表達(dá),可以引起HK-2細(xì)胞凋亡增加,提示Niban可能參與腎小管上皮細(xì)胞凋亡所致的腎間質(zhì)纖維化；3)Niban在腎間質(zhì)纖維化中對HK-2細(xì)胞凋亡的調(diào)節(jié),不通過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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692

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