發(fā)布時間：2018-02-28 20:47

  本文關(guān)鍵詞： 糖尿病腎病 NETs 腎臟纖維化 EMT　出處：《重慶醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的糖尿病腎病是目前導致終末期腎病最主要的原因,但其具體發(fā)病機制尚不明確。中性粒細胞胞外網(wǎng)絡(luò)(neutrophil extracelluar traps,NETs)是中性粒細胞對抗外源性刺激物的一種防御機制。研究發(fā)現(xiàn)NETs在肺纖維化、痛風、哮喘、糖病足等疾病的進程中發(fā)揮了重要作用,但其是否參與了糖尿病腎病的發(fā)生及其機制還未闡明。本研究旨在探索NETs在糖尿病腎病中所發(fā)揮的作用及其機制。方法在動物實驗中,采用一次性腹腔下注射鏈脲酶素(streptozotocin,STZ)150mg/Kg建造糖尿病小鼠模型。常規(guī)喂養(yǎng)32周后收集尿液采用化學酶促法檢測UACR以確定糖尿病腎病模型是否構(gòu)建成功,并處死小鼠后收集腎臟組織。收集血糖正常及糖尿病腎病的腎臟腫瘤患者的瘤旁組織(距腫瘤5cm處)。PAS染色及天狼星紅染色觀察糖尿病腎病的小鼠與人的腎臟結(jié)構(gòu)損傷情況和腎臟纖維化程度,免疫組織化學檢測NETs相關(guān)標志物MPO、Cit-Histone3以及腎小管區(qū)域的上皮間質(zhì)轉(zhuǎn)化情況。在體外實驗中,使用25mmol/L的高糖培養(yǎng)基干預人中性粒細胞,Sytox Green染色觀察NETs產(chǎn)生情況,并采用ds DNA Quantitfy Kit對NETs定量。進一步采用高糖誘導的NETs干預HK2人腎小管上皮細胞24h后,Western Blot檢測E-Cadherin、?-SMA、Snail、ZEB的表達以判斷細胞是否發(fā)生了上皮間質(zhì)轉(zhuǎn)化(epithelial-to-mesenchymal transition,EMT)。結(jié)果1.在小鼠糖尿病腎病模型中,STZ組小鼠UACR較對照組顯著上升(4.421±0.351 mg/ml VS 3.298±0.187 mg/ml),差異具有統(tǒng)計學差異(P0.05)。PAS染色顯示STZ組小鼠的腎臟結(jié)構(gòu)明顯改變,小管區(qū)域基底膜增厚擴張、糖原沉積。天狼星紅染色染色表明STZ組較對照組膠原纖維沉積顯著增多,STZ組腎臟明顯纖維化。免疫熒光染色顯示STZ組NETs標志物MPO、Cit-Histone3沉積明顯增多,免疫組化顯示STZ組小鼠腎小管區(qū)域E-Cadherin表達明顯下調(diào)、?-SMA表達顯著上調(diào),提示腎臟發(fā)生了上皮間質(zhì)轉(zhuǎn)化。2.在人糖尿病腎病組織中,天狼星紅染色顯示腎臟纖維化明顯增加,免疫熒光染色顯示NETs標志物MPO、Cit-Histone3沉積明顯增多,免疫組化顯示腎小管區(qū)域E-Cadherin表達明顯下調(diào)、?-SMA表達顯著上調(diào)。3.在體外實驗中,Sytox-Green熒光染色顯示高糖可誘導中性粒細胞產(chǎn)生NETs。Western Blot顯示高糖誘導的NETs干預人腎小管上皮細胞后E-Cadherin蛋白顯著下調(diào),?-SMA、Snail、ZEB蛋白顯著上調(diào)。結(jié)論NETs在糖尿病腎臟纖維化中發(fā)揮了一定作用,其可能與NETs導致的腎小管上皮間質(zhì)轉(zhuǎn)化有關(guān)。
[Abstract]:Objective Diabetic nephropathy is the main cause of end-stage nephropathy. However, its specific pathogenesis is not clear. Neutrophil extracelluar traps#en0# nets is a defense mechanism of neutrophil against exogenous stimuli. Studies have found that NETs plays an important role in pulmonary fibrosis, gout and asthma. Although glycosylated foot plays an important role in the process of diabetic nephropathy, whether or not it is involved in the pathogenesis of diabetic nephropathy and its mechanism has not been clarified. The purpose of this study was to explore the role and mechanism of NETs in diabetic nephropathy. A diabetic mouse model was established by a single intraperitoneal injection of streptozotocinin (STZ) 150 mg / kg. Urine was collected 32 weeks later to detect UACR by chemical enzymatic method to determine whether the diabetic nephropathy model was successfully constructed. After the mice were killed, the renal tissues were collected. The adjacent tissues of renal tumor patients with normal blood glucose and diabetic nephropathy were collected. The renal structure of diabetic nephropathy mice and human was observed by using pas staining and Sirius red staining at a distance of 5 cm from the tumor. The degree of injury and renal fibrosis, Immunohistochemistry was used to detect the epithelial interstitial transformation of NETs related marker MPO-Cit-Histone3 and renal tubule region. In vitro, 25 mmol / L high glucose medium was used to interfere with human neutrophils Sytox Green staining to observe the production of NETs. NETs was quantified by DS DNA Quantitfy Kit, and E-Cadherinia was detected by Western Blot after 24 h intervention of NETs with high glucose concentration in HK2 human renal tubular epithelial cells. The expression of SMA-Snail-ZEB was used to determine whether the epithelial-to-mesenchymal transition occurred in the cells. Results 1. In the diabetic nephropathy model of mice, the UACR in the STZ group was significantly higher than that in the control group (4.421 鹵0.351 mg/ml vs 3.298 鹵0.187 mg / ml), and the difference was statistically significant (P 0.05). Pas staining showed that the STZ group had a significant increase in UACR. The kidney structure of mice was obviously changed. The thickening and expanding of basement membrane and glycogen deposition in the tubule region. Sirius red staining showed that the deposition of collagen fibers in STZ group was significantly higher than that in control group. Immunofluorescence staining showed that the NETs marker MPO-Cit-Histone3 was significantly increased in STZ group. Immunohistochemical staining showed that the expression of E-Cadherin in the renal tubular region of STZ group was significantly down-regulated. SMA expression was significantly up-regulated, suggesting that epithelial interstitial transformation occurred in the kidney. In human diabetic nephropathy tissues, Sirius red staining showed a marked increase in renal fibrosis, and immunofluorescence staining showed an obvious increase in the deposition of NETs marker MPO-Cit-Histone3. Immunohistochemical staining showed that E-Cadherin expression in renal tubules was significantly down-regulated. In vitro, Sytox-Green fluorescence staining showed that high glucose could induce neutrophils to produce NETs.Western Blot. NETs induced by high glucose significantly down-regulated E-Cadherin protein in human renal tubular epithelial cells. Conclusion NETs may play a role in renal fibrosis in diabetic rats, which may be related to the tubuloepithelial interstitial transformation induced by NETs.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R692.9

【參考文獻】

相關(guān)期刊論文 前1條

1 Ashish N Rao;Nayef M Kazzaz;Jason S Knight;;Do neutrophil extracellular traps contribute to the heightened risk of thrombosis in inflammatory diseases?[J];World Journal of Cardiology;2015年12期

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本文編號：1548881