【摘要】：目的:糖尿病腎病(Diabetic nephropathy,DN)是一種糖尿病(Diabetes mellitus,DM)主要而嚴(yán)重的微血管并發(fā)癥,其發(fā)病機(jī)制錯綜復(fù)雜,其發(fā)生發(fā)展涉及多個因素參與,近年來不少研究表明,氧化應(yīng)激及免疫炎癥反應(yīng)可能參與糖尿病腎損害的不同病理過程并發(fā)揮核心作用。我們的前期研究表明,高糖可誘導(dǎo)氧化應(yīng)激,激活活性氧(Reactive oxygen species,ROS)及核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體蛋白3(Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3,NLRP3)炎癥小體信號通路,誘導(dǎo)活半胱氨酸蛋白酶1(Cysteine-aspartic acid protease1,Caspase1)及促炎細(xì)胞因子白細(xì)胞介素1-β(Interleukine-1 beta,IL-1β)的成熟與分泌。自噬(Autophagy)是一種清除損傷蛋白質(zhì)或細(xì)胞器以維持細(xì)胞內(nèi)穩(wěn)態(tài)的降解過程,通過清除損傷線粒體減少ROS產(chǎn)生而實(shí)現(xiàn)能量回收,可能參與調(diào)節(jié)ROS及下游的NLRP3炎癥小體信號通路,避免過度的免疫炎癥反應(yīng)。然而到目前為止,高糖刺激下腎系膜細(xì)胞(Glomerular mesangial cell,GMC)的自噬水平及調(diào)節(jié)機(jī)制研究尚少,且研究結(jié)果存在爭議,既往研究表明,適當(dāng)增強(qiáng)自噬可能減少氧化應(yīng)激及免疫炎癥反應(yīng),但過度自噬可能引起自噬性細(xì)胞損傷,加重細(xì)胞凋亡。在有限的自噬調(diào)節(jié)機(jī)制研究中,受體結(jié)合絲氨酸/蘇氨酸激酶2(Receptor-interacting serine/threonine-protein kinase 2,RIPK2),一種Nod樣受體(Nod-like receptor,NLR)家族中Nod1/Nod2的重要連接蛋白,被證明既可激活核因子-κB(Nuclear factor-κB,NF-κB)及絲裂原激活蛋白激酶(Mitogen-activated protein kinases,MAPKs)介導(dǎo)的炎癥反應(yīng),還參與Nod1/Nod2介導(dǎo)的自噬反應(yīng),提示RIPK2可能參與調(diào)節(jié)自噬及ROS-NLRP3炎癥小體信號通路,但目前尚缺乏研究探討高糖刺激腎系膜細(xì)胞對RIPK2以及RIPK2與自噬、ROS-NLRP3炎癥小體信號通路的影響,因此本研究通過觀察小鼠腎系膜細(xì)胞中RIPK2、LC3II/I、ROS、Caspase1、IL-1β在不同時間及不同濃度高糖干預(yù)下的表達(dá)變化,同時檢測細(xì)胞培養(yǎng)上清液中IL-1β表達(dá),再通過細(xì)胞轉(zhuǎn)染RIPK2-siRNA干擾RIPK2后再次觀察自噬水平及ROS-NLRP3炎癥小體關(guān)鍵因子的改變,旨在探討高糖對腎系膜細(xì)胞RIPK2、自噬的影響以及自噬對ROS-NLRP3炎癥小體信號通路的調(diào)控作用,為DN的防治提供新的思路。方法:(1)細(xì)胞培養(yǎng)及分組:體外培養(yǎng)小鼠腎小球系膜細(xì)胞(SV40),以高糖作為刺激因素,分為以下三組:(1)正常對照組(Normal control group,NC組):培養(yǎng)基含5.6 mmol/L葡萄糖;(2)滲透壓對照組(Osmotic pressure group,OP組):培養(yǎng)基含5.6 mmol/L葡萄糖+24.4 mmol/L甘露醇。(3)不同高糖濃度干預(yù)組(High glucose group,HG組):培養(yǎng)基分別含10 mmol/L葡萄糖(HG1組)、20 mmol/L葡萄糖(HG2組)、30 mmol/L葡萄糖(HG3組);(2)各組細(xì)胞培養(yǎng)0h、2h、6h、12h、24h、48h、72h后,用Western-blot檢測RIPK2、LC3II/I、Caspase1、IL-1β的蛋白表達(dá);RT-PCR檢測RIPK2、Caspase1、IL-1βmRNA表達(dá);mRFP-GFP-LC3融合蛋白的腺病毒標(biāo)記LC3以觀察自噬流;各組細(xì)胞適時隨機(jī)加入處理因素后裝載2’,7’-二氯熒光黃雙乙酸鹽探針(2’,7’-Dichlorofluorescin Diacetate,DCFH—DA),并用分光光度計檢測細(xì)胞內(nèi)ROS水平變化;ELISA檢測培養(yǎng)上清液IL-1β濃度。(3)RIPK2的siRNA干擾研究:篩選出最佳高糖作用濃度(30mmol/L)和時間(12h)后,細(xì)胞轉(zhuǎn)染RIPK2 siRNA,再次進(jìn)行分組:(1)siNC組:細(xì)胞轉(zhuǎn)染control siRNA加入含5.6 mmol/L葡萄糖培養(yǎng)基培養(yǎng)12h;(2)si RIPK2組:細(xì)胞轉(zhuǎn)染RIPK2siRNA加入含5.6 mmol/L葡萄糖培養(yǎng)基培養(yǎng)12h;(3)HG+siNC組:細(xì)胞轉(zhuǎn)染control siRNA加入含30 mmol/L葡萄糖培養(yǎng)基培養(yǎng)12h;(4)HG+siRIPK2組細(xì)胞轉(zhuǎn)染RIPK2 si RNA加入含30 mmol/L葡萄糖培養(yǎng)基培養(yǎng)12h;再次采用Western-blot、RT-PCR、mRFP-GFP-LC3標(biāo)記+共聚焦顯微鏡、DCFH—DA標(biāo)記+分光光度計及ELISA等方法檢測上述指標(biāo)。(4)統(tǒng)計學(xué)分析:采用SPSS 24.0統(tǒng)計軟件對數(shù)據(jù)進(jìn)行處理分析。符合正態(tài)分布的計量資料以均數(shù)±標(biāo)準(zhǔn)差(?)表示,采用單因素方差分析對多組間均數(shù)進(jìn)行比較,組間多重比較用LSD—t法。我們定義P0.05為差異具有統(tǒng)計學(xué)意義。結(jié)果:(1)與NC組相比,高糖刺激可增加細(xì)胞內(nèi)ROS產(chǎn)量、Caspase1、IL-1β蛋白及mRNA表達(dá),差異均具有統(tǒng)計學(xué)意義(P值均0.05),且具有時間及濃度依賴性。(2)與NC組相比,高糖在短期作用時間內(nèi)(0-12h)可誘導(dǎo)RIPK2、LC3II/I蛋白及RIPK2mRNA表達(dá)(P0.05),且在30 mmol/L高糖作用12h條件下表達(dá)顯著增強(qiáng),超過12h作用時間后RIPK2及LC3II/I表達(dá)下調(diào)(P0.05)。(3)細(xì)胞轉(zhuǎn)染RIPK2 siRNA后成功抑制RIPK2表達(dá)。與siNC組相比,siRIPK2組LC3II/I表達(dá)顯著下調(diào),而細(xì)胞內(nèi)ROS產(chǎn)量、Caspase1及IL-1β蛋白表達(dá)則顯著增加(P0.05)。結(jié)論:(1)高糖可激活小鼠腎系膜細(xì)胞ROS-NLRP3炎癥小體信號通路。(2)高糖對腎小球系膜細(xì)胞自噬具有雙重調(diào)控作用,即高糖刺激可在短期內(nèi)誘導(dǎo)自噬激活而在長期作用時間下內(nèi)起抑制效應(yīng)。(3)RIPK2通過自噬負(fù)性調(diào)節(jié)ROS-NLRP3炎癥小體信號通路,可能參與糖尿病腎病的發(fā)生發(fā)展。
[Abstract]:Objective: Diabetic nephropathy (DN) is a major and serious microvascular complication of diabetes mellitus (DM). Oxidative stress and immune inflammatory responses may be involved in different pathological processes of diabetic renal impairment and play a central role. Our previous studies have shown that high sugar can induce oxidative stress, activate reactive oxygen species (ROS), and bind to oligomeric domain-like receptor protein 3 (NLRP3) inflammatory small body signal pathways, induce cysteine protease 1 (Cystine-aspinal acid protease1, The maturation and secretion of caspase-1 and interleukinine-1beta, IL-1ADH. Autophagy is a process of removing damaged proteins or organelles in order to maintain homeostasis in cells, and energy recovery is achieved by eliminating damage to mitochondria and reducing ROS generation. It is possible to participate in the regulation of the LRP3 inflammatory small signal pathways downstream of ROS and downstream, avoiding excessive immune inflammatory responses. Up to now, the autophagy level and regulation mechanism of renal membrane cells (GMC) under high sugar stimulation is less and the research results are controversial. Previous studies have shown that appropriate enhancement of autophagy may reduce oxidative stress and immune inflammatory response, However, excessive autophagy may cause autophagy cell damage and increase cell apoptosis. Receptor binding serine/ threonine kinase 2 (RIPK2), a Nod-like receptor (NLR) family, and Nod-like receptor (NLR) family, in a limited autophagy mechanism study, has been shown to be both a nuclear factor and a Nuclear factor-LacB, NF-Sepharose B and Mitogen-activated protein kinase (MAPKKs) mediate inflammatory responses, also involved in the Nod1/ No2-mediated autophagy reaction, suggesting that RIPK2 may participate in the regulation of autophagy and ROS-NLRP3 inflammatory cell signaling pathways, In this study, the expression of RIPK2, LC3II/ I, ROS, Caspas1 and IL-1 in renal membrane cells of mice was studied by observing the expression of RIPK2, LC3II/ I, ROS, Caspas1 and IL-1 in renal membrane cells of mice. meanwhile, the expression of IL-1 in the supernatant of the cell culture is detected, and the change of the key factors of the autophagy level and the inflammatory small body of the ROS-NLRP3 is again observed after the RIIPK2-siRNA interferes with RIIPK2 through the cell transfection, and aims to investigate the effect of high sugar on the RIPK2, autophagy of the renal membrane cells and the regulation effect of autophagy on the inflammatory small body signal path of the ROS-NLRP3, It provides a new idea for the prevention and cure of DN. Methods: (1) Cell culture and grouping: In vitro cultured mouse glomerular mesangial cells (NC) were cultured in three groups: (1) Normal control group (NC): medium containing 5. 6 mmol/ L glucose; (2) osmotic pressure group. OP group: The medium contains 5. 6 mmol/ L glucose + 24. 4 mmol/ L mannitol. (3) High glucose concentration intervention group (HG group): The medium contained 10 mmol/ L glucose (HG1 group), 20 mmol/ L glucose (HG2 group), 30 mmol/ L glucose (HG3 group); (2) The cells were cultured for 0h, 2h, 6h, 12h, 24h, 48h and 72h. Protein expression of Caspas1 and IL-1 protein; RT-PCR detection of RIPK2, Caspas1, IL-1, and mRNA expression; mRFP-GFP-LC3 fusion protein adenoviral marker LC3 to observe autophagy; each group of cells was randomly added to the treatment factor and loaded with 2 ', 7 The concentration of IL-1 in cultured supernatant was detected by ELISA. The concentration of IL-1 in cultured supernatant was detected by ELISA. (3) siRNA interference study of RIPK2: After screening out the optimal high sugar concentration (30mmol/ L) and time (12h), the cells were transfected with RIPK2 siRNA, and then grouped: (1) siNC group: the cell transfected control siRNA was added with 5. 6 mmol/ L glucose medium for 12h; (2) the si RIIPK2 group: Cells transfected with RIPK2 siRNA were cultured for 12h with 5. 6 mmol/ L glucose medium; (3) HG + siNC group: the control siRNA was transfected into the cells with 30 mmol/ L glucose medium for 12h; (4) the transfected RIPK2 si RNA of HG + siRIIPK2 group was added with 30 mmol/ L glucose medium for 12h; Western-blot, RT-PCR, mRFP-GFP-LC3 marker + confocal microscopy were used again. The above-mentioned indexes were tested by DCFH and DA labeling + spectrophotometer and ELISA. (4) Statistical analysis: SPSS 10.0 was used to analyze the data. The measurement data conforming to the normal distribution shall be subject to the standard deviation of the normal distribution (? The results showed that the single-factor analysis of variance was used to compare the inter-group comparisons, and the LSD-tt method was used for multiple comparisons among the groups. Results: (1) Compared with NC group, high sugar stimulation could increase ROS production, Caspas1, IL-1, and mRNA expression in cell, and the difference was statistically significant (P <0.05), and had time and concentration dependence. (2) Compared with NC group, high sugar can induce RIPK2, LC3II/ I protein and RIIPK2 mRNA expression (P0.05) in short-term action time (0-12h), and the expression of RIPK2 and LC3II/ I is down-regulated under the action of 30 mmol/ L high sugar for 12h (P0.05). (3) After transfected with RIPK2 siRNA, the expression of RIPK2 was successfully inhibited. Compared with siNC group, the expression of LC3II/ I was down-regulated in siRIIPK2 group, while in cell ROS production, the expression of caspase-1 and IL-1 was significantly increased (P0.05). Conclusion: (1) High sugar can activate the rat renal membrane cell ROS-NLRP3 inflammatory small body signal pathway. (2) High sugar has a double regulation effect on the autophagy of glomerular mesangial cells, that is, high sugar stimulation can induce autophagy in the short term and play an inhibitory effect in long-term action time. (3) RIIPK2 may be involved in the development of diabetic nephropathy by regulating ROS-NLRP3 inflammatory small body signal pathway from autophagy.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R692.9

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相關(guān)期刊論文 前1條

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本文編號：2287995