单核细胞caspase活性与肥胖和2型糖尿病的关系

发布时间：2018-04-23 01:20

本文选题：肥胖 + 2型糖尿病　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:肥胖是一种表现为体内脂肪堆积过多和(或)分布异常、体重增加的慢性代谢性疾病。糖尿病是以血糖升高为特征的代谢性疾病,90%以上为2型糖尿病(T2DM)。肥胖和T2DM的共同特征为胰岛素抵抗,而胰岛素抵抗与系统性慢性低度炎症密切相关。近年来研究显示,肥胖和T2DM个体的脂肪组织中M1型(炎性)巨噬细胞增多。半胱氨酸天冬氨酸特异性蛋白酶(caspase)是细胞程序性死亡过程中的关键酶,炎性caspase通过激活炎症小体,促进炎性细胞因子IL-1β和IL-18的成熟和释放。本研究通过检测血浆中单核细胞caspase活性,分析其与体重指数BMI、腰围、血糖、血脂、血中炎性细胞因子浓度之间的相关性,同时体外高糖刺激人THP-1单核细胞,检测单核细胞活性,探讨单核细胞caspase与肥胖和T2DM的关系,为肥胖和T2DM的发病和治疗机制新视角。内容:第一部分:招募健康人和T2DM病人,测量身高、体重、腰围,计算BMI,根据BMI和腰围分为3组:正常组、T2DM组、T2DM合并肥胖组。每名志愿者各抽取2ml EDTA抗凝血和2ml分离胶促凝血,检测空腹血糖(FPG)、糖化血红蛋白(HbA1c)、甘油三酯(TG)和总胆固醇(TC)水平。提取抗凝血中的单个核细胞,检测单核细胞caspase活性,分析单核细胞caspase活性与以上各指标的相关性第二部分:收集正常组、T2DM组、T2DM合并肥胖组分离胶促凝血,检测血清中IL-1β、TNF-α、脂多糖(LPS)、游离脂肪酸(NEFA)水平,并分析单核细胞caspase活性与以上各指标的相关性。第三部分:体外培养人THP-1单核细胞株,首先用含33mM葡萄糖的1640培养THP-1,分别培养12小时、24小时、36小时、48小时,检测单核细胞caspase活性;然后分别用含25mM、33mM葡萄糖的1640培养12小时和24小时,检测细胞上清液中乳酸脱氢酶(LDH)水平,以含11mM葡萄糖1640培养的THP-1单核细胞作为对照,进一步验证高血糖对于单核细胞活性的影响。方法:招募健康人和T2DM病人共37人,测量身高、体重、腰围,计算BMI,根据BMI和腰围分为正常组、T2DM组、T2DM合并肥胖组,生化全自动分析仪检测血液中FPG、TG、TC,糖化血红蛋白分析仪检测HbA1c,流式细胞术检测单核细胞caspase活性,ELISA检测血清IL-1β、TNF-α、LPS水平,游离脂肪酸试剂盒检测血清中NEFA水平。在体外用含33mM葡萄糖的1640培养THP-1,分别培养12小时、24小时、36小时、48小时,流式细胞术检测单核细胞caspase活性,然后分别用含25mM、33mM葡萄糖的1640培养THP-1单核细胞12小时和24小时,细胞毒性试剂盒检测细胞上清液中LDH。结果:第一部分结果显示:T2DM合并肥胖组总单核细胞、炎性单核细胞以及经典单核细胞caspase活性均显著高于正常组,总单核细胞caspase活性与BMI、腰围显著正相关,与FPG、HbA1c、TG、TC不相关。第二部分结果显示:T2DM合并肥胖组IL-1β水平显著高于正常组和T2DM组,T2DM组NEFA水平显著高于正常组,但T2DM合并肥胖组NEFA水平与正常组无显著差异。总单核细胞caspase活性、炎性单核细胞caspase活性均与IL-1β显著正相关,与TNF-α、LPS、NEFA不相关。第三部分结果显示:与含11mM葡萄糖的1640培养的THP-1单核细胞相比,高糖的培养条件并未影响单核细胞的caspase活性以及细胞上清液中LDH水平。结论:肥胖和T2DM可能在外周血单核细胞caspase活性升高中发挥协同促进作用。
[Abstract]:Objective: obesity is a chronic metabolic disease characterized by excessive fat accumulation and (or) distribution and increasing body weight. Diabetes is a metabolic disease characterized by elevated blood sugar, more than 90% is type 2 diabetes (T2DM). Obesity and T2DM are characterized by insulin resistance, and insulin resistance and systemic chronic low inflammation are closely related to insulin resistance. In recent years, studies have shown that M1 (inflammatory) macrophages in fat tissues and T2DM individuals have increased. Cysteine aspartate specific protease (caspase) is the key enzyme in the process of programmed cell death. Inflammatory caspase promotes the maturation and release of inflammatory cytokines IL-1 beta and IL-18 by activating inflammatory cells. The relationship between the caspase activity of monocyte in plasma and the concentration of body mass index (BMI), waist circumference, blood sugar, blood lipid and inflammatory cytokines in blood was analyzed, and the activity of THP-1 mononuclear cells was stimulated by high glucose in vitro, and the relationship between the mononuclear cell caspase and obesity and T2DM was explored, and the pathogenesis of obesity and T2DM and the pathogenesis of T2DM were analyzed. New perspective of treatment mechanism. Content: first part: recruit healthy people and T2DM patients, measure height, weight, waist circumference, calculate BMI, divide into 3 groups according to BMI and waist circumference: normal group, T2DM group, T2DM combined obesity group. Each volunteer takes 2ml EDTA anticoagulant and 2ml separation gel prothrombin, fasting blood glucose (FPG), glycated hemoglobin (HbA1c), glycerin Three ester (TG) and total cholesterol (TC) level. Extract the mononuclear cells in anticoagulant, detect the caspase activity of mononuclear cells, analyze the correlation between the caspase activity of mononuclear cells and the above indexes, to collect the normal group, T2DM group, T2DM combined with the obesity group and promote the coagulation of blood, and detect the serum IL-1 beta, TNF- a, lipopolysaccharide (LPS), free fat. The level of acid (NEFA), and the correlation between the caspase activity of mononuclear cells and the above indexes. Third: in vitro culture, human THP-1 mononuclear cell line was cultured with 33mM glucose 1640 for 12 hours, 24 hours, 36 hours and 48 hours respectively to detect the caspase activity of mononuclear cells, and then with 25mM, 33mM glucose 1640 respectively. The level of lactate dehydrogenase (LDH) in the cell supernatant was detected for 12 hours and 24 hours, and the THP-1 mononuclear cells cultured with 11mM glucose 1640 were used as control. The effect of hyperglycemia on the activity of mononuclear cells was further verified. Methods: 37 people were recruited and T2DM patients were recruited to measure height, weight, waist circumference, BMI, BMI and waistline. For the normal group, the T2DM group and the T2DM group, the biochemical full automatic analyzer was used to detect the FPG, TG, TC, and the glycosylated hemoglobin analyzer to detect the HbA1c, the flow cytometry was used to detect the caspase activity of mononuclear cells. ELISA was used to detect the serum IL-1 beta, TNF- alpha and LPS levels. The free fatty acid kit was used to detect the level of the serum NEFA level. In vitro, 1 of the glucose containing glucose was used. 640 THP-1 was cultured for 12 hours, 24 hours, 36 hours and 48 hours respectively. Flow cytometry was used to detect the caspase activity of mononuclear cells. Then, THP-1 mononuclear cells were incubated with 25mM and 33mM glucose for 12 hours and 24 hours respectively. The cytotoxic kit was used to detect the LDH. results in the cell supernatant. The first part showed that the T2DM combined with the obesity group was total. The activity of monocyte, inflammatory mononuclear cell and classical monocyte caspase were significantly higher than that of normal group. The caspase activity of total monocyte was significantly correlated with BMI, waist circumference, and not related to FPG, HbA1c, TG, TC. The second results showed that the level of IL-1 beta in T2DM combined obesity group was significantly higher than that in normal group and T2DM group, and NEFA level in T2DM group was significantly higher than that in normal group. The NEFA level in the T2DM combined obesity group was not significantly different from that in the normal group. The total monocyte caspase activity and the caspase activity of the inflammatory monocytes were positively correlated with the IL-1 beta, and were not related to TNF- alpha, LPS, NEFA. The third part showed that the high sugar culture conditions were not compared with the THP-1 mononuclear cells containing 11mM glucose of 1640. Caspase activity of mononuclear cells and the level of LDH in cell supernatant. Conclusion: obesity and T2DM may play a synergistic role in promoting high school of caspase activity in peripheral blood mononuclear cells.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R589.2

【参考文献】