發(fā)布時(shí)間：2018-02-23 23:29

  本文關(guān)鍵詞： n-3 PUFAs 糖尿病腎病 fat-1轉(zhuǎn)基因小鼠 IL-1β　出處：《寧波大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:糖尿病腎病是糖尿病最嚴(yán)重的并發(fā)癥,也是糖尿病患者死亡的主要原因之一。研究發(fā)現(xiàn)n-3多不飽和脂肪酸(n-3 PUFAs)及下游代謝產(chǎn)物有抗炎和抑制氧化應(yīng)激作用,但是n-3 PUFAs能否夠改善糖尿病腎病及其作用機(jī)制還有待闡明。本研究利用fat-1轉(zhuǎn)基因小鼠建立糖尿病腎病模型,探索內(nèi)源性n-3 PUFAs拮抗糖尿病腎病的效果及其作用機(jī)理,為今后利用n-3 PUFAs膳食預(yù)防和治療糖尿病腎病提供理論依據(jù)。方法:fat-1雜合子基因小鼠和野生型小鼠雜交,出生4周的小鼠采用PCR方法鑒定fat-1基因小鼠和野生型小鼠,然后各自隨機(jī)分成2組。給4組小鼠喂飼富含n-6PUFAs的紅花籽油,7周后注射鏈脲佐菌素(STZ,60mg/kg.d),連續(xù)注射5天誘導(dǎo)糖尿病模型。然后定期稱量小鼠體重、測(cè)量小鼠血糖、收集小鼠代謝物(尿液量、飲水量、糞便重量)。注射STZ 12周后小鼠血糖達(dá)到30 mmol/L左右,且產(chǎn)生明顯的蛋白尿。處死小鼠,收集小鼠血漿測(cè)定生化指標(biāo),ELISA法測(cè)定脂肪組織培養(yǎng)液IL-1β含量。腎臟和胰腺組織固定、切片制作、病理學(xué)觀察及氣相色譜法測(cè)定腎臟脂肪酸含量及液相色譜-紫外-串聯(lián)質(zhì)譜測(cè)定脂肪酸的衍生物含量。Western Blot法檢測(cè)腎臟中IL-1β及相關(guān)蛋白的表達(dá)。結(jié)果:1、采用PCR-凝膠電泳法鑒定小鼠基因,獲得fat-1雜合子基因小鼠(fat-1小鼠)和野生型小鼠(WT小鼠)。2、建立糖尿病腎病模型:(1)注射STZ后,72h后小鼠血糖≥16.7 mmol/L,12周后小鼠血糖達(dá)到30 mmol/L左右;注射STZ小鼠體重與正常組相比,間行性下降(P0.05)。(2)注射STZ的WT小鼠(WT+STZ小鼠)飲水量、尿液更多,尿蛋白程度更嚴(yán)重。3、fat-1小鼠中內(nèi)源性n-3 PUFAs對(duì)腎臟結(jié)構(gòu)影響:WT小鼠和fat-1小鼠腎臟結(jié)構(gòu)完整,WT+STZ小鼠腎小球基底膜增厚,腎小球系膜擴(kuò)增,腎小管破壞嚴(yán)重;和WT+STZ小鼠相比,fat-1+STZ小鼠腎臟損傷相對(duì)較輕。4、內(nèi)源性n-3 PUFAs減輕糖尿病腎病的作用機(jī)理:(1)與fat-1+STZ小鼠相比,WT+STZ小鼠血漿中BUN、Scr和脂肪組織培養(yǎng)液中IL-1β含量增加(P0.05)。(2)小鼠腎臟中脂肪酸分布情況:與WT小鼠和WT+STZ小鼠比較,fat-1小鼠和fat-1+STZ小鼠腎臟中n-3 PUFAs及衍生脂類介質(zhì)增加,n-6/n-3 PUFAs比例降低(P0.05)。(3)腎臟組織蛋白免疫印跡結(jié)果顯示:與WT小鼠和fat-1小鼠相比,WT+STZ和fat-1+STZ小鼠caspase-1、cleaved IL-1β、p-NF-κB表達(dá)上調(diào);與fat-1+STZ小鼠比較,WT+STZ組小鼠cleaved IL-1β表達(dá)增加和SOD1表達(dá)減少。結(jié)論:本研究發(fā)現(xiàn),一方面,fat-1小鼠體內(nèi)增加的內(nèi)源性n-3 PUFAs上調(diào)超氧化物歧化酶SOD1的表達(dá),表達(dá)增加的SOD1能抑制體內(nèi)氧化應(yīng)激,進(jìn)而阻止炎癥小體NLRP3活化,減少促炎因子cleaved IL-1β的產(chǎn)生,從而改善STZ誘導(dǎo)的糖尿病腎病。另一方面,內(nèi)源性增加的n-3 PUFAs上調(diào)E-cardherin表達(dá),下調(diào)N-cardherin表達(dá),減輕STZ誘導(dǎo)的腎臟纖維化過程,從而改善糖尿病腎病。因此,我們的研究結(jié)果為利用n-3 PUFAs延緩糖尿病腎病提供理論依據(jù),對(duì)今后利用含n-3 PUFAs膳食預(yù)防和治療糖尿病腎病都具有重要意義。
[Abstract]:Objective: diabetic nephropathy is the most serious complication of diabetes and one of the main causes of death in diabetic patients. It has been found that n-3 polyunsaturated fatty acid n-3PUFAsand its downstream metabolites have anti-inflammatory and anti-oxidative stress effects. However, whether n-3 PUFAs can improve diabetic nephropathy and its mechanism remains to be clarified. In this study, we used fat-1 transgenic mice to establish diabetic nephropathy model and to explore the antagonistic effect and mechanism of endogenous n-3 PUFAs on diabetic nephropathy. In order to provide a theoretical basis for the prevention and treatment of diabetic nephropathy with n-3 PUFAs diet in the future. Methods the mice with fat-1 gene and wild-type mice were hybridized with the heterozygous gene of 1: fat-1. The mice of 4 weeks old were identified by PCR method, and the mice with fat-1 gene and wild-type mice were identified by PCR method. Then they were randomly divided into two groups. The mice in 4 groups were fed with n-6 PUFAs rich in safflower seed oil for 7 weeks. After 7 weeks, streptozotocin (STZ) 60 mg / kg 路dl was injected to induce diabetic model for 5 days, then the mice were weighed regularly and blood glucose was measured. After 12 weeks of STZ injection, the blood sugar of the mice reached about 30 mmol/L, and the mice produced significant proteinuria. IL-1 尾 content in adipose tissue culture medium was determined by Elisa. Kidney and pancreas tissue were fixed, and sections were made. Pathological observation and gas chromatographic determination of fatty acid content in kidney and determination of derivative content of fatty acid in kidney by liquid chromatography-UV-tandem mass spectrometry. Western Blot method was used to detect the expression of IL-1 尾 and related protein in kidney. Electrophoretic identification of mouse genes, The fat-1 heterozygous gene mice were obtained. The diabetic nephropathy model was established by using fat-1 heterozygous gene mice and wild-type mice. The model of diabetic nephropathy was established. The blood glucose of mice was more than 16.7 mmol 路L ~ (-1) after injection of STZ 72 h later, the blood glucose of mice was about 30 mmol/L after 12 weeks, and the body weight of mice injected with STZ was about 30 mmol/L compared with the normal group. The effect of endogenous n-3 PUFAs on renal structure in WT STZ mice injected with STZ, urine and urine protein was more serious. The glomerular basement membrane was thickened in the mice with intact kidney structure of WT STZ mice and the intact kidney structure of fat-1 mice, and the effect of endogenous n-3 PUFAs on the renal structure was observed in the mice with FT-1.The results showed that the glomerular basement membrane of WT STZ mice was thicker than that of the control mice. Glomerular Mesangial amplification, renal tubule destruction; Compared with WT STZ mice, the kidney damage of STZ mice was lighter than that of WT STZ mice, and the mechanism of endogenous n-3 PUFAs in alleviating diabetic nephropathy was: 1) compared with fat-1 STZ mice, the contents of BUNN SCR in plasma and IL-1 尾 in adipose tissue culture medium increased in fat-1 STZ mice. Distribution of fatty acids in kidney: compared with WT mice and WT STZ mice, n-3 PUFAs and derivative lipid mediators in kidney of fat-1 and fat-1 STZ mice increased the ratio of n-6 / n-3 PUFAs and decreased the ratio of n-6 / n-3 PUFAs.) the results of Western blot of kidney tissue showed that: compared with WT mice, the ratio of n-3 PUFAs and derived lipids in kidney increased. Compared with fat-1 mice, the expression of caspase-1 and IL-1 尾 -p-NF- 魏 B was up-regulated in WT STZ and fat-1 STZ mice. Compared with fat-1 STZ mice, cleaved IL-1 尾 expression and SOD1 expression decreased in WT STZ mice. Conclusion: in this study, it was found that endogenous n-3 PUFAs increased SOD1 expression of superoxide dismutase in fat-1 mice. The increased expression of SOD1 could inhibit oxidative stress in vivo, thus inhibit the activation of NLRP3 in inflammatory corpuscles and decrease the production of cleaved IL-1 尾, thus improving STZ induced diabetic nephropathy. On the other hand, the endogenous increase of n-3 PUFAs upregulated the expression of E-cardherin. By down-regulating the expression of N-cardherin, the process of renal fibrosis induced by STZ can be alleviated, thus improving diabetic nephropathy. Therefore, our findings provide a theoretical basis for the use of n-3 PUFAs to delay diabetic nephropathy. It is of great significance for the prevention and treatment of diabetic nephropathy with n-3 PUFAs diet in the future.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R692.9

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