【摘要】：目的：糖尿病腎�。╠iabetic nephropathy,DN）是糖尿病常見(jiàn)且特有的微血管并發(fā)癥之一，其危害巨大，然而目前具體的發(fā)病機(jī)制仍不清楚。國(guó)內(nèi)外大量研究表明，炎癥是糖尿病腎病發(fā)生發(fā)展的中心環(huán)節(jié)，核因子-κB（NF-κB）信號(hào)是介導(dǎo)糖尿病腎�。―N）炎癥的重要通路。最新研究表明，作為模式識(shí)別受體的NOD樣受體（NOD1）在NF-κB信號(hào)通路的調(diào)控激活中起著重要作用。細(xì)胞外刺激可經(jīng)過(guò)NOD1-RICK-IκB等信號(hào)分子激活NF-κB信號(hào)通路，介導(dǎo)炎癥反應(yīng)。NOD1介導(dǎo)的NF-κB信號(hào)通路是否參與了糖尿病腎病的發(fā)病尚未見(jiàn)相關(guān)文獻(xiàn)報(bào)道。因此，本研究采用大鼠腎小球系膜細(xì)胞(HBZY-1)進(jìn)行體外培養(yǎng)，高糖、脂多糖（lipopolysaccharide,LPS）作為刺激因子，NOD1受體抑制劑（ML130）作為阻斷劑，分別采用western-blot和RT-PCR檢測(cè)NOD1、RICK、IκB、NF-κB等的蛋白及mRNA表達(dá),旨在探討NOD1-RICK-NF-κB信號(hào)在糖尿病腎病炎癥信號(hào)中的作用，為糖尿病腎病防治提供新的理論基礎(chǔ)。方法：1.體外培養(yǎng)大鼠腎小球系膜細(xì)胞，分別給予葡萄糖、LPS以及NOD1的抑制劑（ML130）干預(yù)，分成以下7組：①正常對(duì)照組(NC組)：培養(yǎng)基含葡萄糖濃度為5.6mmol/L；②高糖組（HG1,HG2和HG3組）：培養(yǎng)基所含葡萄糖濃度分別為10mmol/L、20mmol/L和30mmol/L；③滲透壓對(duì)照組：培養(yǎng)基含葡萄糖5.6mmol/L和24.4mmol/L的甘露醇；④LPS組（LPS1組,LPS2和LPS3組）：培養(yǎng)基中LPS的濃度分別為1μg/L、5μg/L和10μg/L；⑤高糖+LPS組：培養(yǎng)基含30mmol/L葡萄糖和5μg/L LPS；⑥高糖+ML130組：含30mmol/L葡萄糖培養(yǎng)基中預(yù)先加入30μmol/L ML130（NOD1受體阻斷劑）；⑦LPS+ML130組：含5μg/L LPS的培養(yǎng)基中預(yù)先加入30μmol/LML130；2.分別將各組細(xì)胞培養(yǎng)一定時(shí)間后，，采用免疫印跡(Western-blotting)和RT-PCR技術(shù)檢測(cè)NOD1、RICK、IκB、NF-κB的蛋白及基因表達(dá);3.統(tǒng)計(jì)學(xué)分析：應(yīng)用SPSS17.0統(tǒng)計(jì)軟件分析數(shù)據(jù)。所有的數(shù)據(jù)都用均數(shù)±標(biāo)準(zhǔn)差（x±s）來(lái)表示，各組之間的數(shù)據(jù)比較采用單因素方差分析，組內(nèi)兩兩比較采用LSD t檢驗(yàn)，檢驗(yàn)水準(zhǔn)α=0.05，P0.05表明有統(tǒng)計(jì)學(xué)意義。結(jié)果：1.與NC組比較，不同濃度的高糖作用不同時(shí)間均可上調(diào)大鼠腎小球系膜細(xì)胞的NOD1、RICK、NF-κB p65蛋白和mRNA的表達(dá)、下調(diào)IκBα表達(dá)（均P0.05），提示高糖呈時(shí)間和濃度依賴性刺激NOD1-RICK-NF-κB信號(hào)；2.與NC組比較，不同濃度的LPS作用不同時(shí)間可以上調(diào)系膜細(xì)胞NOD1、RICK、NF-κB蛋白及mRNA的表達(dá)，下調(diào)IκBα表達(dá)（均P0.05）,提示LPS能激活NOD1-RICK-NF-κB的信號(hào)；3.與高糖組和LPS組相比，高糖+LPS組可增強(qiáng)NOD1、RICK、NF-κB蛋白和mRNA表達(dá)，抑制IκBα表達(dá)，說(shuō)明高糖和LPS能協(xié)同刺激NOD1-RICK-NF-κB信號(hào)。4.與高糖組和LPS組相比，預(yù)先加入ML130可阻斷高糖和LPS誘導(dǎo)的NOD1、RICK、NF-κB的高表達(dá)和IκBα的低表達(dá)（均P0.05），提示高糖和LPS是通過(guò)NOD1受體激活NF-κB信號(hào)通路。結(jié)論：高糖及LPS通過(guò)NOD1受體激活系膜細(xì)胞RICK-NF-κB炎癥信號(hào)通路，NOD1介導(dǎo)的NF-κB信號(hào)通路可能參與了糖尿病腎病發(fā)生。
[Abstract]:Objective: diabetic nephropathy (diabetic nephropathy DN) is one of the common and unique microvascular complications of diabetes mellitus. A large number of studies at home and abroad have shown that inflammation is the central link in the occurrence and development of diabetic nephropathy, and nuclear factor- 魏 B (NF- 魏 B) signal is an important pathway to mediate the inflammation of diabetic nephropathy (DN). Recent studies have shown that NOD like receptors (NOD1), as pattern recognition receptors, play an important role in the regulation and activation of NF- 魏 B signaling pathway. Extracellular stimulation can activate NF- 魏 B signaling pathway via signal molecules such as NOD1-RICK-I 魏 B, and whether NF- 魏 B signaling pathway mediated by inflammatory response. NOD1-mediated NF- 魏 B signal pathway is involved in the pathogenesis of diabetic nephropathy has not been reported. In this study, rat glomerular Mesangial cells (HBZY-1) were cultured in vitro. High glucose and lipopolysaccharide (LPS) were used as stimulator for inhibitor of NOD1 receptor (ML130). Western-blot and RT-PCR were used to detect the expression of protein and mRNA in rat glomerular Mesangial cells (HBZY-1). To explore the role of NOD1-RICK-NF- 魏 B signal in the inflammatory signal of diabetic nephropathy and to provide a new theoretical basis for the prevention and treatment of diabetic nephropathy. Method 1: 1. Rat glomerular Mesangial cells were cultured in vitro and were treated with glucose lipopolysaccharide and NOD1 inhibitor (ML130) respectively. They were divided into the following 7 groups: normal control group (NC group): the concentration of glucose was 5.6 mmol / L in culture medium; 2 High glucose group (HG1HG2 and HG3 group): the glucose concentration in the medium was 10 mmol / L, 20 mmol / L and 30 mmol / L / L, respectively. The osmotic pressure control group: mannitol 4 LPS group (LPS1 group, LPS2 and LPS3 group) containing glucose 5.6mmol/L and 24.4mmol/L: the concentration of LPS in the medium was 1 渭 g / L 5 渭 g / L and 10 渭 g / L, respectively; 5 High glucose LPS group: medium containing 30mmol/L glucose and 5 渭 g / L LPS6 high glucose ML130 group: 30mmol/L glucose medium with 30 渭 mol/L ML130 (NOD1 receptor blocker) 7 LPs ML130 group: medium containing 5 渭 g / L LPS with 30 渭 mol / L LML130 + 2. After the cells were cultured for a certain time, the protein and gene expression of I 魏 B NF- 魏 B were detected by Western blotting (Western-blotting) and RT-PCR. Statistical analysis: SPSS17.0 statistical software was used to analyze the data. All the data were represented by mean 鹵standard deviation (x 鹵s). The data of each group were compared with single factor ANOVA, and the intra-group comparison was performed with LSD t test. The result is 1: 1. Compared with NC group, different concentrations of high glucose could up-regulate the expression of NOD1-RICKB p65 protein and mRNA in rat glomerular Mesangial cells and down-regulate the expression of I 魏 B 偽 (P0.05), suggesting that high glucose stimulated NOD1-RICK-NF- 魏 B signal 2 in a time-and concentration-dependent manner. Compared with NC group, the expression of NOD1-RICK-NF- 魏 B protein and mRNA in Mesangial cells could be upregulated by different concentrations of LPS at different time, and I 魏 B 偽 expression was down-regulated (P0.05), suggesting that LPS could activate the signal of NOD1-RICK-NF- 魏 B. Compared with high glucose group and LPS group, high glucose LPS group could enhance the expression of NOD1-RICK- 魏 B protein and mRNA and inhibit the expression of I 魏 B 偽, indicating that high glucose and LPS could co-stimulate NOD1-RICK-NF- 魏 B signal .4. Compared with high glucose group and LPS group, the high expression of NF- 魏 B and the low expression of I 魏 B 偽 induced by high glucose and LPS were blocked by ML130, suggesting that high glucose and LPS activated NF- 魏 B signaling pathway through NOD1 receptor. Conclusion: high glucose and LPS may be involved in the pathogenesis of diabetic nephropathy through NOD1 receptor activated Mesangial cells RICK-NF- 魏 B inflammatory signaling pathway and NOD1-mediated NF- 魏 B signaling pathway.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.2;R692.9

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