内源性n-3多不饱和脂肪酸拮抗糖尿病肾病的机理研究

发布时间：2018-02-23 23:29

  本文关键词： n-3 PUFAs 糖尿病肾病 fat-1转基因小鼠 IL-1β　出处：《宁波大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:糖尿病肾病是糖尿病最严重的并发症,也是糖尿病患者死亡的主要原因之一。研究发现n-3多不饱和脂肪酸(n-3 PUFAs)及下游代谢产物有抗炎和抑制氧化应激作用,但是n-3 PUFAs能否够改善糖尿病肾病及其作用机制还有待阐明。本研究利用fat-1转基因小鼠建立糖尿病肾病模型,探索内源性n-3 PUFAs拮抗糖尿病肾病的效果及其作用机理,为今后利用n-3 PUFAs膳食预防和治疗糖尿病肾病提供理论依据。方法:fat-1杂合子基因小鼠和野生型小鼠杂交,出生4周的小鼠采用PCR方法鉴定fat-1基因小鼠和野生型小鼠,然后各自随机分成2组。给4组小鼠喂饲富含n-6PUFAs的红花籽油,7周后注射链脲佐菌素(STZ,60mg/kg.d),连续注射5天诱导糖尿病模型。然后定期称量小鼠体重、测量小鼠血糖、收集小鼠代谢物(尿液量、饮水量、粪便重量)。注射STZ 12周后小鼠血糖达到30 mmol/L左右,且产生明显的蛋白尿。处死小鼠,收集小鼠血浆测定生化指标,ELISA法测定脂肪组织培养液IL-1β含量。肾脏和胰腺组织固定、切片制作、病理学观察及气相色谱法测定肾脏脂肪酸含量及液相色谱-紫外-串联质谱测定脂肪酸的衍生物含量。Western Blot法检测肾脏中IL-1β及相关蛋白的表达。结果:1、采用PCR-凝胶电泳法鉴定小鼠基因,获得fat-1杂合子基因小鼠(fat-1小鼠)和野生型小鼠(WT小鼠)。2、建立糖尿病肾病模型:(1)注射STZ后,72h后小鼠血糖≥16.7 mmol/L,12周后小鼠血糖达到30 mmol/L左右;注射STZ小鼠体重与正常组相比,间行性下降(P0.05)。(2)注射STZ的WT小鼠(WT+STZ小鼠)饮水量、尿液更多,尿蛋白程度更严重。3、fat-1小鼠中内源性n-3 PUFAs对肾脏结构影响:WT小鼠和fat-1小鼠肾脏结构完整,WT+STZ小鼠肾小球基底膜增厚,肾小球系膜扩增,肾小管破坏严重;和WT+STZ小鼠相比,fat-1+STZ小鼠肾脏损伤相对较轻。4、内源性n-3 PUFAs减轻糖尿病肾病的作用机理:(1)与fat-1+STZ小鼠相比,WT+STZ小鼠血浆中BUN、Scr和脂肪组织培养液中IL-1β含量增加(P0.05)。(2)小鼠肾脏中脂肪酸分布情况:与WT小鼠和WT+STZ小鼠比较,fat-1小鼠和fat-1+STZ小鼠肾脏中n-3 PUFAs及衍生脂类介质增加,n-6/n-3 PUFAs比例降低(P0.05)。(3)肾脏组织蛋白免疫印迹结果显示:与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表达上调;与fat-1+STZ小鼠比较,WT+STZ组小鼠cleaved IL-1β表达增加和SOD1表达减少。结论:本研究发现,一方面,fat-1小鼠体内增加的内源性n-3 PUFAs上调超氧化物歧化酶SOD1的表达,表达增加的SOD1能抑制体内氧化应激,进而阻止炎症小体NLRP3活化,减少促炎因子cleaved IL-1β的产生,从而改善STZ诱导的糖尿病肾病。另一方面,内源性增加的n-3 PUFAs上调E-cardherin表达,下调N-cardherin表达,减轻STZ诱导的肾脏纤维化过程,从而改善糖尿病肾病。因此,我们的研究结果为利用n-3 PUFAs延缓糖尿病肾病提供理论依据,对今后利用含n-3 PUFAs膳食预防和治疗糖尿病肾病都具有重要意义。
[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.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

【相似文献】

中国期刊全文数据库 前10条

1 王静,阮芸,徐冬娥,谭擎缨;多项指标联检对诊断早期糖尿病肾病的意义[J];浙江临床医学;2000年04期

2 王玉新,李大启,李公宝,徐琴君;糖尿病肾病患者血清可溶性白细胞介素6受体检测[J];上海免疫学杂志;2001年02期

3 宫雅南,刘冬年,熊玉冰,黄伟文,庄万江;糖尿病足患者糖尿病视网膜病变分析[J];广东医学;2001年06期

4 顾芹,宋守君,李向阳,尤传一;胰激肽原酶肠溶片对早期糖尿病肾病的防治作用[J];中国临床药学杂志;2001年06期

5 曹爱华 ,王瑛 ,李翔;杏丁治疗早期糖尿病肾病疗效观察[J];辽宁实用糖尿病杂志;2001年03期

6 ;中药治疗糖尿病肾病新进展[J];中国中医药信息杂志;2001年04期

7 张桂茹;糖尿病肾病85例临床分析[J];陕西医学杂志;2002年02期

8 侯建明 ,林凤辉 ,张超群;46例2型糖尿病肾病与糖尿病自主神经病变的关系分析[J];福建医药杂志;2002年04期

9 陈文霖;糖尿病饮食[J];医疗保健器具;2002年Z1期

10 孙力,许玲;2型糖尿病患者糖尿病足的危险因素分析[J];山东医药;2002年35期

中国重要会议论文全文数据库 前10条

1 张星;许筠;苏建平;张军;程立志;翟晓丽;;糖尿病肾病的临床疗效对比观察[A];第十九次全国中医肾病学术交流会论文汇编[C];2006年

2 杨家茂;;糖尿病肾病防治琐谈[A];全国第二届中医中西医结合肾脏病临床进展学术研讨会论文集[C];2007年

3 丁耀耿;郝桂霞;;糖尿病肾病临床分析[A];全国第二届中医中西医结合肾脏病临床进展学术研讨会论文集[C];2007年

4 张文铠;王志伏;王雪;孙大朋;;糖尿病肾病的治疗现状[A];中华中医药学会第二十一届全国中医肾病学术会议论文汇编（下）[C];2008年

5 倪青;;糖尿病肾病的中西医结合研究[A];第六届全国中西医结合肾脏病学术会议论文汇编[C];2000年

6 孙怡;李健红;宗红燕;;黄芪桂枝五物汤加味治疗糖尿病肾病16例[A];第六届全国中西医结合肾脏病学术会议论文汇编[C];2000年

7 叶军;;糖尿病肾病在儿童时期的早期干预[A];中华医学会第六次全国内分泌学术会议论文汇编[C];2001年

8 杜旭昶;孙志红;闫春芳;刘彩虹;;疏糖丹治疗2型糖尿病50例临床分析[A];第六次中国中西医结合糖尿病学术会议论文汇编[C];2002年

9 于世家;任平;马丽佳;李小娟;郑曙琴;武明东;刘自力;薛丽辉;;糖尿病住院患者1344例回顾性分析[A];第六次中国中西医结合糖尿病学术会议论文汇编[C];2002年

10 郝效槐;魏玫都;崔立俊;;中西并蓄治疗糖尿病肾病[A];第七次中国中西医结合糖尿病学术会议论文汇编[C];2004年

中国重要报纸全文数据库 前10条

1 本报记者 向佳;糖尿病中医药防治项目立足社区[N];中国中医药报;2011年

2 特约记者 鲁海燕;逾八成公众存在糖尿病高危因素[N];家庭医生报;2013年

3 马明愈;现代生活方式导致 糖尿病发病率迅速上升[N];中国妇女报;2005年

4 省立医院内分泌科主任医师 侯建明;糖尿病肾病的防治[N];福建科技报;2004年

5 王文绢 范军星;世界糖尿病日关注焦点:糖尿病并发症[N];健康报;2003年

6 主持人 向红丁博士;糖尿病肾病须早防早治[N];人民政协报;2002年

7 华悦;预防糖尿病，，从减肥开始[N];上海中医药报;2004年

8 刘冬梅;肥胖糖尿病第一诱因[N];天津日报;2004年

9 刘燕玲;首部中医专病指南定下糖尿病治则[N];健康报;2007年

10 崔昕;中药防治糖尿病肾病有进展[N];健康时报;2006年

中国博士学位论文全文数据库 前10条

1 王晓杰;组蛋白去乙酰化酶4特异性促进糖尿病肾病足细胞损伤[D];山东大学;2015年

2 张永;MiR-346在抗TGF-β信号途径介导的糖尿病肾病发生和发展中的作用机制[D];武汉大学;2015年

3 魏凤江;高尿酸血症、2型糖尿病及糖尿病微血管病变的群体遗传学研究[D];天津医科大学;2015年

4 孙士杰;胱抑素C对2型糖尿病视网膜病变预测价值研究[D];山东大学;2015年

5 龙泓竹;益气养阴通络散结方防治早期糖尿病肾病的临床及实验研究[D];北京中医药大学;2016年

6 姜e

本文编号：1528025