高糖诱导的NETs在糖尿病肾病中的作用及机制研究

发布时间：2018-02-28 20:47

本文关键词： 糖尿病肾病 NETs 肾脏纤维化 EMT　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的糖尿病肾病是目前导致终末期肾病最主要的原因,但其具体发病机制尚不明确。中性粒细胞胞外网络(neutrophil extracelluar traps,NETs)是中性粒细胞对抗外源性刺激物的一种防御机制。研究发现NETs在肺纤维化、痛风、哮喘、糖病足等疾病的进程中发挥了重要作用,但其是否参与了糖尿病肾病的发生及其机制还未阐明。本研究旨在探索NETs在糖尿病肾病中所发挥的作用及其机制。方法在动物实验中,采用一次性腹腔下注射链脲酶素(streptozotocin,STZ)150mg/Kg建造糖尿病小鼠模型。常规喂养32周后收集尿液采用化学酶促法检测UACR以确定糖尿病肾病模型是否构建成功,并处死小鼠后收集肾脏组织。收集血糖正常及糖尿病肾病的肾脏肿瘤患者的瘤旁组织(距肿瘤5cm处)。PAS染色及天狼星红染色观察糖尿病肾病的小鼠与人的肾脏结构损伤情况和肾脏纤维化程度,免疫组织化学检测NETs相关标志物MPO、Cit-Histone3以及肾小管区域的上皮间质转化情况。在体外实验中,使用25mmol/L的高糖培养基干预人中性粒细胞,Sytox Green染色观察NETs产生情况,并采用ds DNA Quantitfy Kit对NETs定量。进一步采用高糖诱导的NETs干预HK2人肾小管上皮细胞24h后,Western Blot检测E-Cadherin、?-SMA、Snail、ZEB的表达以判断细胞是否发生了上皮间质转化(epithelial-to-mesenchymal transition,EMT)。结果1.在小鼠糖尿病肾病模型中,STZ组小鼠UACR较对照组显著上升(4.421±0.351 mg/ml VS 3.298±0.187 mg/ml),差异具有统计学差异(P0.05)。PAS染色显示STZ组小鼠的肾脏结构明显改变,小管区域基底膜增厚扩张、糖原沉积。天狼星红染色染色表明STZ组较对照组胶原纤维沉积显著增多,STZ组肾脏明显纤维化。免疫荧光染色显示STZ组NETs标志物MPO、Cit-Histone3沉积明显增多,免疫组化显示STZ组小鼠肾小管区域E-Cadherin表达明显下调、?-SMA表达显著上调,提示肾脏发生了上皮间质转化。2.在人糖尿病肾病组织中,天狼星红染色显示肾脏纤维化明显增加,免疫荧光染色显示NETs标志物MPO、Cit-Histone3沉积明显增多,免疫组化显示肾小管区域E-Cadherin表达明显下调、?-SMA表达显著上调。3.在体外实验中,Sytox-Green荧光染色显示高糖可诱导中性粒细胞产生NETs。Western Blot显示高糖诱导的NETs干预人肾小管上皮细胞后E-Cadherin蛋白显著下调,?-SMA、Snail、ZEB蛋白显著上调。结论NETs在糖尿病肾脏纤维化中发挥了一定作用,其可能与NETs导致的肾小管上皮间质转化有关。
[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 卤0.351 mg/ml vs 3.298 卤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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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