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类型 应用研究 预答辩日期 2017-12-01
开始(开题)日期 2016-06-12 论文结束日期 2017-10-19
地点 公共卫生学院三楼会议室 论文选题来源 国家自然科学基金项目     论文字数 5.4 (万字)
题目 不同来源ω-3多不饱和脂肪酸对2型糖尿病合并血脂异常人群糖脂代谢的影响及机制研究
主题词 ω-3多不饱和脂肪酸,2型糖尿病,糖脂代谢,胰岛素抵抗,TLR4/MyD88
摘要 糖尿病已成为当前威胁全球人类健康最重要的慢性非传染性疾病之一。2型糖尿病(Type 2 diabetes mellitus,T2DM)患者不仅存在糖代谢紊乱,还多伴有血脂异常。近年来,ω-3多不饱和脂肪酸(Polyunsaturated fatty acid, PUFA)对心血管疾病的保护效应成为医学及营养学界关注的热点。膳食中ω-3 PUFA分为动物性来源和植物性来源。动物性来源以二十碳五烯酸(Eicosapentaenoic acid, EPA)和二十二碳六烯酸(Docosahexenoic acid, DHA)为代表。植物性来源以ɑ-亚麻酸(Alpha-linolenic acid, ALA)为代表。目前对动物性来源ω-3 PUFA与T2DM发病风险及糖脂代谢的人群干预研究相对较多,但结果尚存争议,植物性来源ω-3 PUFA的研究相对较少,需要深入开展不同来源ω-3 PUFA对T2DM合并血脂异常人群糖脂代谢影响的研究,并探讨其可能的作用机制。 本研究采用动物性来源ω-3 PUFA(鱼油)、植物性来源ω-3 PUFA(紫苏油)、动植物性来源兼有的ω-3 PUFA(鱼油+亚麻油),对T2DM合并血脂异常人群进行为期6个月的干预,综合比较不同来源ω-3 PUFA对其糖脂代谢的影响。同时通过高脂饲料喂养C57BL/6小鼠和MyD88-/-小鼠构建胰岛素抵抗(Insulin resistance, IR)模型,分别研究动物性来源ω-3 PUFA(鱼油)、植物性来源ω-3 PUFA(紫苏油)经TLR4/MyD88依赖性途径与TLR4/MyD88非依赖途径调控IR的机制。研究内容分为三个部分: 第一部分 不同来源ω-3 PUFA对T2DM合并血脂异常人群糖脂代谢的影响 目的:探讨不同来源ω-3 PUFA对T2DM合并血脂异常人群糖脂代谢的影响。方法:于2017年2月~2017年3月在江苏省宜兴市官林医院糖尿病专科门诊招募180例T2DM合并血脂异常患者,随机分为鱼油组、紫苏油组和鱼油+亚麻油组,每组60人,每日分别给予3g鱼油胶囊(含EPA 1.14g,DHA 1.44g)、3g紫苏油胶囊(含ALA 2.04g)和3g鱼油+亚麻油胶囊(含EPA 0.63g,DHA 0.36g,ALA 0.84g),持续时间6个月。在基线、干预3个月、干预6个月时采集空腹血,使用Mindray BS-800全自动生化分析仪测定血糖、甘油三酯(Triglyceride, TG)、总胆固醇(Total cholesterol, TC)、高密度脂蛋白胆固醇(High density lipoprotein-cholesterol, HDL-C)、低密度脂蛋白胆固醇(Low density lipoprotein-cholesterol, LDL-C)、载脂蛋白A1(Apolipoprotein A1, Apo A1)、载脂蛋白B(Apolipoprotein B, Apo B)、脂蛋白(a)[Lipoprotein(a), Lp(a)]、游离脂肪酸(Free fatty acid, FFA)、谷丙转氨酶、谷草转氨酶、肌酐、尿酸、尿素氮、总蛋白、白蛋白和球蛋白,使用Audicom AC6601全自动糖化血红蛋白分析仪测定糖化血红蛋白(Glycated haemoglobin, HbAlc),使用Roche cobas e 602电化学发光分析仪测定C肽和胰岛素。结果:共有156人完成6个月的干预研究,其中鱼油组54人,紫苏油组52人,鱼油+亚麻油组50人。与基线相比,鱼油组、紫苏油组和鱼油+亚麻油组C肽、胰岛素、稳态模型评估胰岛素抵抗指数、TG、TC、非HDL-C、Apo A1均降低(P<0.05),鱼油组HDL-C升高(P<0.05),紫苏油组和鱼油+亚麻油组血糖、HbAlc、Apo B、Lp(a)均降低(P<0.05),鱼油+亚麻油组FFA降低(P<0.05)。与紫苏油组和鱼油+亚麻油组相比,鱼油组干预3个月TG下降较为明显(P<0.05),HDL-C升高接近有统计学差异(P=0.055)。结论:动物性来源ω-3 PUFA、植物性来源ω-3 PUFA和动植物性来源兼有的ω-3 PUFA在改善T2DM合并血脂异常人群糖代谢方面的作用相似,动物性来源ω-3 PUFA降低T2DM合并血脂异常人群TG的作用优于植物性来源ω-3 PUFA和动植物性来源兼有的ω-3 PUFA。 第二部分 不同来源ω-3 PUFA经TLR4/MyD88依赖性途径调控IR的机制研究 目的:探讨不同来源ω-3 PUFA经TLR4/MyD88依赖性途径调控IR的机制。方法:将6周龄无特定病原体(Specified pathogens free, SPF)级健康雄性C57BL/6小鼠适应性喂养1周后,按体重随机分为正常对照组、模型对照组、鱼油组和紫苏油组,每组10只。正常对照组给予AIN-93M普通饲料喂养,模型对照组、鱼油组和紫苏油组给予D12451高脂饲料喂养。鱼油组和紫苏油组每日按0.5g/kg bw分别给予鱼油(每100g鱼油含EPA 38g、DHA 48g)和紫苏油(每100g紫苏油含ALA 68g)灌胃,持续4周。实验结束时,小鼠禁食不禁水12h~14h,摘眼球取血后颈椎脱臼处死,分离附睾白色脂肪组织。使用Beckman DxC800全自动生化分析仪测定血糖、TG、TC、HDL-C、LDL-C,使用Merck Millipore MAGPIX多因子检测试剂盒测定胰岛素、单核细胞趋化蛋白1(Monocyte chemotactic protein-1, MCP-1)、白细胞介素6(Interleukin-6, IL-6)、肿瘤坏死因子α(Tumor necrosis factor-α, TNF-α)、瘦素和抵抗素。采用RT-PCR和Western Blot方法检测白色脂肪组织TLR4、MyD88、TRAF6、IKKβ、NF-κB p65、RIP1、IRF3的mRNA和蛋白表达水平,同时对白色脂肪组织进行苏木精-伊红染色。结果:与正常对照组相比,模型对照组血糖、胰岛素、TG、TC、HDL-C、LDL-C、非HDL-C、LDL-C/HDL-C、IL-6、TNF-α和瘦素均上升(P<0.05),而且200倍光镜下、25mm2内白色脂肪细胞数减少(P<0.05),白色脂肪细胞间质炎细胞浸润程度加深。经鱼油和紫苏油干预后,血糖、胰岛素、TG、TC、HDL-C、LDL-C、非HDL-C和IL-6均下降(P<0.05),而且200倍光镜下、25mm2内白色脂肪细胞数增多(P<0.05),鱼油组LDL-C低于紫苏油组(P<0.05)。由RT-PCR结果与Western Blot结果可见,模型对照组较正常对照组TLR4、MyD88、TRAF6、IKKβ、NF-κB p65、RIP1、IRF3的mRNA和蛋白表达均增加(P<0.05),经鱼油和紫苏油干预后,TLR4、MyD88、TRAF6、IKKβ、NF-κB p65的mRNA和蛋白表达均较模型对照组下降(P<0.05)。鱼油组MyD88、IKKβ相对mRNA和蛋白表达均低于紫苏油组(P<0.05)。结论:动物性来源ω-3 PUFA和植物性来源ω-3 PUFA均可以改善高脂饮食诱导的C57BL/6小鼠IR和血脂异常,其机制涉及TLR4/MyD88依赖性途径。 第三部分 不同来源ω-3 PUFA经TLR4/MyD88非依赖性途径调控IR的机制研究 目的:探讨不同来源ω-3 PUFA经TLR4/MyD88非依赖性途径调控IR的机制。方法:将8周龄SPF级健康雄性MyD88-/-小鼠适应性喂养1周后,按体重随机分为正常对照组、模型对照组、鱼油组和紫苏油组,每组6只。正常对照组给予AIN-93M普通饲料喂养,模型对照组、鱼油组和紫苏油组给予D12451高脂饲料喂养。鱼油组和紫苏油组每日按0.5g/kg bw分别给予鱼油(每100g鱼油含EPA 38g、DHA 48g)和紫苏油(每100g紫苏油含ALA 68g)灌胃,持续4周。实验结束时,小鼠禁食不禁水12h~14h,摘眼球取血后颈椎脱臼处死,分离附睾白色脂肪组织。使用Beckman DxC800全自动生化分析仪测定血糖、TG、TC、HDL-C、LDL-C,使用Merck Millipore MAGPIX多因子检测试剂盒测定胰岛素、MCP-1、IL-6、TNF-α、瘦素和抵抗素。采用RT-PCR和Western Blot方法检测白色脂肪组织TLR4、RIP1、IRF3、NF-κB p65的mRNA和蛋白表达水平,同时对白色脂肪组织进行苏木精-伊红染色。结果:与正常对照组相比,模型对照组血糖、胰岛素、TC、HDL-C、瘦素和抵抗素均上升(P<0.05)。模型对照组、鱼油组和紫苏油组血糖、胰岛素、TC、HDL-C、MCP-1、IL-6、TNF-α、瘦素和抵抗素无统计学差异。正常对照组、模型对照组、鱼油组和紫苏油组200倍光镜下、25mm2内白色脂肪细胞数无统计学差异。由RT-PCR结果与Western Blot结果可见,模型对照组较正常对照组TLR4、RIP1、IRF3、NF-κB p65的mRNA和蛋白表达均增加(P<0.05),经鱼油和紫苏油干预后,RIP1、IRF3、NF-κB p65的mRNA和蛋白表达均无明显变化。结论:动物性来源ω-3 PUFA与植物性来源ω-3 PUFA均不能通过TLR4/MyD88非依赖性途径改善高脂饮食诱导的MyD88-/-小鼠IR和血脂异常。
英文题目 Effects of ω-3 polyunsaturated fatty acids from different sources on glucolipid metabolism and its mechanism in type 2 diabetic patients with dyslipidemia
英文主题词 ω-3 polyunsaturated fatty acid, type 2 diabetes mellitus, glucolipid metabolism, insulin resistance, TLR4/MyD88
英文摘要 Diabetes mellitus is currently one of the most important chronic non-communicable diseases worldwide. People with type 2 diabetes mellitus (T2DM) not only have glucose metabolism dysfunction, but also often express dyslipidemia. In recent years, the protective effects of ω-3 polyunsaturated fatty acid (PUFA) on cardiovascular disease have become a hot spot of concern in the medicine and nutrition communities. Dietary ω-3 PUFA can be classified into animal-derived and plant-derived ω-3 PUFA. Animal-derived ω-3 PUFA is represented by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Alpha-linolenic acid (ALA) represents the plant-derived ω-3 PUFA. At present, there are relatively large number of controversial researches in animal-derived ω-3 PUFA, but relatively small number of those in plant-derived ω-3 PUFA regarding T2DM risk and intervention research. Therefore, there is a great need for deeply understanding the effects of ω-3 PUFA from different sources on glucose and lipid metabolism in type 2 diabetic patients with dyslipidemia and its mechanism. This study aimed to determine the effects of fish oil-derived ω-3 PUFA, perilla oil-derived ω-3 PUFA, fish oil mixed with linseed oil-derived ω-3 PUFA on glucolipid metabolism in type 2 diabetic patients with dyslipidemia. Meanwhile, C57BL/6 mice and MyD88-/- mice fed on high fat diet were used to reveal the mechanism of insulin resistance (IR) via TLR4/MyD88 dependent way and TLR4/MyD88 independent way. Part 1: Effects of ω-3 polyunsaturated fatty acids from different sources on glucolipid metabolism in type 2 diabetic patients with dyslipidemia Objective: (1) To determine the effects of ω-3 polyunsaturated fatty acids from different sources on glucolipid metabolism in type 2 diabetic patients with dyslipidemia. Methods: We recruited participants from the diabetes clinic at the Guanlin hospital in Yixing city, Jiangsu province from February 2017 to March 2017. 180 subjects were randomly assigned to 3g/day fish oil (FO, containing 1.14g EPA and 1.44g DHA), 3g/day perilla oil (PO, containing 2.04g ALA), or 3g/day fish oil mixed with linseed oil (FLO, containing 0.63g EPA , 0.36g DHA and 0.84g ALA) for 6 months. Fasting venous blood sample was obtained from each study subject at baseline, after 3 and 6 months of intervention. Serum glucose, triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein A1 (Apo A1), apolipoprotein B (Apo B), Lipoprotein(a) [Lp(a)], free fatty acid (FFA), glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, creatinine, uric acid, urea nitrogen, total protein, albumin and globulin were determined by automatic biochemical analyzer (Mindray, BS-800). Glycated haemoglobin (HbAlc) was determined by chromatography (Audicom, AC6601). C peptide and insulin were determined by chemiluminescence (Roche, cobas e 602). Results: 156 subjects completed the final follow-up after 6 months (FO, 54 subjects; PO, 52 subjects; FLO, 50 subjects). C peptide, insulin, homeostasis model assessment-insulin resistance (HOMA-IR), TG, TC, non HDL-C and Apo A1 in FO, PO and FLO were significantly lower than that at the baseline (P<0.05). Glucose, HbAlc, Apo B and Lp(a) in PO and FLO were significantly lower than that at the baseline (P<0.05). FFA in FLO was significantly lower than that at the baseline (P<0.05). Compared with PO and FLO, TG in FO significantly decreased (P<0.05) after 3 months. There was a trend for HDL-C to increase after fish oil supplementation (P=0.055). Conclusion: The effects of ω-3 PUFA from different sources on glucose metabolism in type 2 diabetic patients with dyslipidemia are similar. TG-lowering effect of animal-derived ω-3 PUFA in type 2 diabetic patients with dyslipidemia shows an advantage over plant-derived ω-3 PUFA and animal mixed with plant-derived ω-3 PUFA. Part 2: Effects of ω-3 polyunsaturated fatty acids from different sources on insulin resistance via TLR4/MyD88 dependent way in C57BL/6 mice Objective: To investigate the effects of ω-3 PUFA from different sources on IR via TLR4/MyD88 dependent way in C57BL/6 mice. Methods: Six-week-old specified pathogens free male C57BL/6 mice were randomly divided into 4 groups (n=10 in each group) after a week of acclimization period according to body weight: (1) normal control (NC) group was fed normal AIN-93M diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium, (2) model control (MC) group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium, (3) fish oil (FO) group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw fish oil (38g EPA and 48g DHA per 100g), and (4) perilla oil (PO) group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw perilla oil (68g ALA per 100g). After administration of ω-3 PUFAs for 4 weeks, the animals were fasted for 12-14 hours and then sacrificed with sodium pentothal. Blood and epididymal adipose tissue samples were collected for further analyses. Serum glucose, TG, TC, HDL-C and LDL-C were determined by automatic biochemical analyzer (Beckman, DxC800). Serum insulin, monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), leptin and resistin were measured using MILLIPLEX? map kits (Merck Millipore). The expression of TLR4, MyD88, TRAF6, IKKβ, NF-κB p65, RIP1 and IRF3 in epididymal adipose tissue were examined by RT-PCR and western-blot. The epididymal adipose tissue was stained with eosin-hematoxylin for pathological examination. Results: Serum glucose, insulin, TG, TC, HDL-C, LDL-C, non HDL-C, LDL-C/HDL-C, IL-6 TNF-α and leptin were significantly increased in MC than those in NC (P<0.05). However, significantly lower glucose, insulin, TG, TC, HDL-C, LDL-C, non HDL-C and IL-6 were observed in FO and PO, as opposed to MC (P<0.05). The histological examination of epididymal adipose tissue in MC revealed decrease of adipocyte counts (P<0.05) and serious inflammatory cell infiltration. These histopathological changes were ameliorated by fish oil and perilla oil supplementation (P<0.05). Both RT-PCR and western-blot showed that the expression of TLR4, MyD88, TRAF6, IKKβ, NF-κB p65, RIP1 and IRF3 were significantly increased in MC than that in NC (P<0.05). However, significantly lower expression of TLR4, MyD88, TRAF6, IKKβ and NF-κB p65 were observed in FO and PO, as opposed to MC (P<0.05). Conclusion: These findings indicate that animal-derived ω-3 PUFA and plant-derived ω-3 PUFA could be used to prevent IR and dyslipidemia via TLR4/MyD88 dependent way in C57BL/6 mice. Part 3: Effects of ω-3 polyunsaturated fatty acids from different sources on insulin resistance via TLR4/MyD88 independent way in MyD88-/- mice Objective: To investigate the effects of ω-3 PUFA from different sources on IR via TLR4/MyD88 independent way in MyD88-/- mice. Methods: Eight-week-old specified pathogens free male MyD88-/- mice were randomly divided into 4 groups (n=6 in each group) after a week of acclimization period according to body weight: (1) normal control (NC) group was fed normal AIN-93M diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium, (2) model control (MC) group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium, (3) fish oil (FO) group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw fish oil (38g EPA and 48g DHA per 100g), and (4) perilla oil (PO) group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw perilla oil (68g ALA per 100g). After administration of ω-3 PUFAs for 4 weeks, the animals were fasted for 12-14 hours and then sacrificed with sodium pentothal. Blood and epididymal adipose tissue samples were collected for further analyses. Serum glucose, TG, TC, HDL-C and LDL-C were determined by automatic biochemical analyzer (Beckman, DxC800). Serum insulin, MCP-1, IL-6, TNF-α, leptin and resistin were measured using MILLIPLEX? map kits (Merck Millipore). The expression of TLR4, RIP1, IRF3 and NF-κB p65 in epididymal adipose tissue were examined by RT-PCR and western-blot. The epididymal adipose tissue was stained with eosin-hematoxylin for pathological examination. Results: Serum glucose, insulin, TC, HDL-C, leptin and resistin were significantly increased in MC than those in NC (P<0.05). Serum glucose, insulin, TC, HDL-C, MCP-1, IL-6, TNF-α, leptin and resistin were not significantly different in FO and PO, as opposed to MC. The adipocyte counts in MC, FO and PO had no differences from NC. Both RT-PCR and western-blot showed that the expression of TLR4, RIP1, IRF3 and NF-κB p65 were significantly increased in MC than that in NC (P<0.05). However, the expression of TLR4, MyD88, TRAF6, IKKβ and NF-κB p65 in FO and PO had no differences from MC. Conclusion: These findings indicate that animal-derived ω-3 PUFA and plant-derived ω-3 PUFA could not be used to prevent IR and dyslipidemia via TLR4/MyD88 independent way in MyD88-/- mice.
学术讨论
主办单位时间地点报告人报告主题
中国营养学会 2015年5月16日 北京国家会议中心 李铎 膳食胆固醇:一个多次被误解的老化合物
东南大学公共卫生学院 2015年5月26日 东南大学公共卫生学院 王锋 The fatty acids and risk of type 2 diabetes
东南大学公共卫生学院 2015年12月26日 东南大学公共卫生学院 王锋 掷出窗外:面对食品安全危机,你应有的态度
上海胡锦华健康教育促进中心 2016年5月23日 上海世纪皇冠假日酒店 康景轩 A tale of omega-3: from fat to fat-1
东南大学公共卫生学院 2016年10月10日 东南大学公共卫生学院 王锋 Vitamin E and Health
东南大学公共卫生学院 2017年5月17日 东南大学公共卫生学院 王锋 Efficacy of fish oil-derived n-3 PUFA on inflammatory and oxidative stress markers in a randomized controlled trial of type 2 diabetic patients with abdominal obesity
中国营养学会 2017年5月22日 北京国家会议中心 李光伟 高危人群糖尿病预防---药物还是生活方式干预?
中国营养学会 2017年9月14日 无锡君来湖滨饭店 孙桂菊 全球营养素补充剂相关政策及我国营养素补充剂共识的制定
     
学术会议
会议名称时间地点本人报告本人报告题目
东南大学研究生校庆学术报告会 2016年5月17日 东南大学公共卫生学院 Efficacy of fish oil-derived n-3 polyunsaturated fatty acids on glucose and lipid metabolism in a randomized controlled trial of type 2 diabetes mellitus patients with abdominal obesity
International Conference on Advances in Human Nutrition. Food Science 2016年6月26日 Toronto Efficacy of fish oil-derived n-3 polyunsaturated fatty acids on inflammatory and oxidative stress markers in a randomized controlled trial of type 2 diabetes mellitus patients with abdominal obesity
     
代表作
论文名称
Treatment for 6 months with fish oil-derived n-3 polyunsaturated fatty acids has neutral effects on
Antioxidant activities of aqueous extracts from 12 Chinese edible flowers in vitro and in vivo
Consumption of a liquid high-fat meal increases triglycerides but decreases high-density lipoprotein
Effects of a liquid high-fat meal on postprandial lipid metabolism in type 2 diabetic patients with
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
陈瑞 正高 教授 博导 东南大学
粱戈玉 正高 教授 博导 东南大学
许茜 正高 教授 博导 东南大学
冯晴 正高 教授 博导 南京医科大学
袁宝君 正高 主任医师 硕导 江苏省疾病预防控制中心
      
答辩秘书信息
姓名职称工作单位备注
杨立刚 其他 讲师 东南大学