【作者】 梁琳琅；

【导师】 祝之明；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 背景和目的:代谢综合征(Metabolic syndrome,MS)是一组以中心性肥胖、胰岛素抵抗(Insulin resisitin,IR)、高血压、血脂异常、糖调节受损或2型糖尿病为主要表现的临床症候群,其病理生理基础为IR。MS发病机制尚不十分清楚,近年来的研究显示,中心性肥胖、脂肪组织内分泌功能失调可能在其中发挥重要作用,相关研究已成为本领域热点,备受关注。脂肪组织能够分泌多种脂肪细胞因子参与调节机体各系统和组织的功能,影响胰岛素敏感性、血压水平、内皮功能、纤溶活动及炎症反应等,参与多种重要病理生理过程。脂联素(Adiponectin,APN)是脂肪细胞特异分泌的蛋白激素,研究表明,其具有抗IR、抗MS、抗动脉粥样硬化和抗炎症等作用,有望成为以IR为特征的、肥胖相关性疾病如MS、2型糖尿病、心血管病等的预测性诊断指标和新的治疗研究靶点。视黄醇结合蛋白4(Retinol binding protein 4,RBP4)是新近发现的脂肪细胞因子,研究表明,其参与糖代谢调节,并诱导IR。目前尚完全不清楚RBP4在人体内的病理生理调节机制,也未明了RBP4在MS发病机制中的作用及其影响因素。本课题从临床角度入手,观察MS患者血清APN、RBP4水平的临床特征,探讨其在MS中可能的作用及其与MS各组分的关系。先前的研究已提示,血清APN、RBP4水平均存在显著的性别差异,机制尚不清楚,一般认为与性激素有关,有待进一步研究。为明确相关机制,本课题分别进行了相关指标的血清水平、离体脂肪组织及体外脂肪细胞蛋白表达及其相关因素的研究,并对上述推论进行探讨。研究分为三个部分:⑴MS患者血清APN、RBP4水平变化的临床特征及与性激素内环境状态的相关性研究,明确APN和RBP4在MS中可能的作用及其与MS各组分的关系、性别差异特点,以及与垂体-性腺轴多种性激素、性激素结合蛋白(Sex hormone binding globulin,SHBG)血浆水平的关系;⑵MS患者腹内(网膜)脂肪及皮下脂肪APN及其受体1(Adiponectin receptor 1,Adipo R1)、RBP4蛋白表达的研究,明确中心性肥胖时上述指标在不同性别、不同部位脂肪组织的表达特点,探讨其与血清ANP、RBP4及性激素水平的关系;⑶性激素对3T3-L1前脂肪细胞和脂肪细胞APN、Adipo R1、RBP4蛋白表达的影响,及性激素对3T3-L1前脂肪细胞分化成熟和成脂的影响的研究,初步探讨MS患者APN和RBP4表达存在性别差异与性激素相关性的分子机制。材料方法:整个研究分临床和基础实验两部分。临床研究是对临床研究对象一般资料及有关指标血清水平进行观察;基础实验是针对临床研究对象腹内及皮下脂肪组织和3T3-L1前脂肪细胞及诱导分化为成熟脂肪细胞进行的研究。1.将临床研究对象分为MS组、非代谢综合征(NMS)组和健康对照(C)组,测量体脂参数、血压,检测一般生化指标,测定糖代谢指标、脂代谢指标、炎症指标、血清APN和RBP4等水平,问卷调查吸烟、饮酒习惯。探讨MS患者血清APN、RBP4水平变化的临床特征及其影响因素。2.将临床研究对象分为(中心性)肥胖组(n=50)和非肥胖组(n=34),测量一般临床指标,测定血清APN、RBP4、性激素及SHBG等水平。探讨MS患者血清APN、RBP4的性别差异特征与性激素内环境状态的关系。3.采用免疫荧光法定性测定腹内和皮下脂肪组织APN、Adipo R1、RBP4的表达和分布;蛋白免疫印迹(Western blot)方法测量腹内和皮下脂肪组织APN、Adipo R1、RBP4的蛋白表达,进行男女组间及MS和C组亚组间的比较;探讨腹内和皮下脂肪组织APN、Adipo R1、RBP4表达与血清水平之间的关系及其和性激素内环境状态的关系。4.采用油红染色、光镜观察10-10M(低生理浓度)、10-8M(生理浓度)、10-6M(超生理浓度)雌二醇(E2)和3×10-10M(低生理浓度)、3×10-8 M(生理浓度)、3×10-6M(超生理浓度)睾酮(T)干预下3T3-L1前脂肪细胞和脂肪细胞中脂滴聚集情况,并计算油红染色脂滴面积占总面积的百分比,比较不同浓度、不同种类性激素对3T3-L1前脂肪细胞和脂肪细胞成脂作用的差异。5.3T3-L1前脂肪细胞实验分组:未诱导性激素干预组、诱导同时性激素干预组和诱导成熟时性激素干预组,同时分为上述不同浓度干预亚组,以及C组;采用Western blot方法测量3T3-L1前脂肪细胞和脂肪细胞APN、Adipo R1、RBP4的蛋白表达;探讨性激素对3T3-L1细胞APN、Adipo R1、RBP4蛋白表达的影响,初步探讨APN、RBP4存在显著性别差异的分子机制。结果:1.临床研究显示:⑴MS组血清RBP4水平显著增加,血浆APN水平显著降低,并存在性别差异:男性血清RBP4水平显著高于女性,男性血清APN水平显著低于女性。⑵血清RBP4水平与性别和BMI独立相关;血清APN水平与WC和血清视RBP4水平独立相关。⑶MS组血清T和E2激素水平呈与性别相反表达,血清SHBG水平显著降低,性激素内环境变化与血清RBP4、APN水平可能相关。2.定性观察人体腹内和皮下脂肪组织APN、Adipo R1、RBP4均有表达,分布以细胞膜为主。3.离体脂肪组织研究显示:⑴脂肪组织APN、Adipo R1、RBP4的蛋白表达均存在性别差异。⑵MS组网膜脂肪组织APN表达显著降低,男性显著低于女性;女性比男性皮下脂肪组织APN表达显著增高。⑶女性MS组网膜脂肪组织Adipo R1表达显著降低;女性MS组网膜脂肪组织Adipo R1表达显著高于男性MS组;女性网膜和皮下脂肪组织Adipo R1表达均显著高于男性;男性皮下脂肪组织Adipo R1表达显著高于腹内脂肪组织。⑷男性MS组网膜脂肪组织RBP4表达显著降低,并显著低于女性MS组;男性皮下脂肪组织RBP4表达显著低于女性。女性皮下脂肪组织RBP4表达显著高于腹内脂肪组织。4.3T3-L1细胞培养后油红染色、比较成脂情况:⑴未诱导性激素干预组,72h时E210-8M干预组成脂显著强于C组,其他各组与C组差异无显著意义。⑵诱导同时性激素干预组,与C组相比,不同浓度E2和T干预组成脂情况与C组差异无显著意义。⑶诱导成熟时性激素干预组,与C组相比,E210-10M干预组、E210-8M干预组和T 3×10-8 M干预组成脂情况显著增强,而T 3×10-10M干预组显著减弱。5.3T3-L1细胞培养:⑴未诱导性激素干预组,与对照组相比,E210-10M干预组和不同浓度T干预组细胞APN表达均显著增高,T 3×10-10M干预组、T 3×10-6M干预组细胞Adipo R1表达均显著增高,T 3×10-10M干预组细胞RBP4表达显著降低;⑵诱导同时性激素干预组,T 3×10-8 M干预组细胞APN表达显著增高,T 3×10-8 M干预组和T 3×10-6M干预组细胞Adipo R1表达均显著增高,E210-6M干预组细胞Adipo R1表达均显著降低,T 3×10-8 M干预组和T 3×10-6M干预组细胞RBP4表达均显著增高,E210-6M干预组细胞RBP4表达显著降低;⑶诱导成熟时性激素干预组,E210-8M干预组和T 3×10-8 M干预组细胞APN表达均显著增高,不同浓度T干预组细胞Adipo R1表达均显著增高,E210-10M干预组、E210-8M干预组和不同浓度T干预组细胞RBP4表达均显著增高。结论:1.MS患者血清RBP4水平显著增加、APN水平显著降低,血清RBP4、APN水平均与多种MS组分密切相关,且与APN与RBP4独立相关。2.MS患者体内存在性激素内环境紊乱,主要表现为性别相关性性激素下调趋势和血清SHBG的显著降低;血清RBP4、APN水平均存在显著的性别差异,与血清性激素及SHBG水平密切相关。3.人体腹内和皮下脂肪组织APN、Adipo R1、RBP4均有表达,分布以细胞膜为主,且均存在性别差异。⑴MS患者网膜脂肪组织APN和Adipo R1表达特点及性别差异与APN血清水平特征相似,表明APN在MS时血浆水平显著降低,是由于腹内脂肪细胞APN分泌显著减少所致,且APN对脂肪组织Adipo R1表达可能具有正向调节作用,从而进一步影响APN生物学效应的发挥。⑵MS患者腹内脂肪组织RBP4表达特点及性别差异与血清RBP4水平特征不一致,表明MS时及男性血循环中RBP4增高并非来源于腹内脂肪组织,可能与机体代谢因素和其他器官组织功能的影响相关。4.生理浓度、低生理浓度的E2和生理浓度的T均对诱导成熟的脂肪细胞具有促进成脂的作用,而低生理浓度的T则有抑制作用。5.性激素对3T3-L1前脂肪细胞和脂肪细胞APN、Adipo R1、RBP4的蛋白表达均有影响,不同浓度的E2、T的干预作用不同,可能提示MS时性激素内环境变化在一定程度上可能影响脂肪组织APN、RBP4的表达。综上所述,MS时血清和脂肪组织APN、AdipoR1、RBP4的异常变化在一定程度上受性激素的调节,这种调节可能参与了MS组分形成、糖脂代谢紊乱等病理生理过程。更多还原

【Abstract】 Background and Objective:Metabolic syndrome (MS) is a group of metabolic risk factors characterized by central obesity, insulin resistance, hypertension, dyslipidemia, and impaired glucose tolerance or type 2 diabetes mellitus. Insulin resistance is thought to be the main pathophysiologic basis of metabolic syndrome, in which underlying pathogenesis involved remains unclear. Recently, a body of reports demonstrates that central obesity and endocrine dysfunction of adipose tissue might pay an important role in the pathogenesis of metabolic syndrome, and related issues have become the focus of research in this field. Many kinds of adipocytokines secreted by adipocytes are implicated in homeostasis of the whole body, including regulation of many pathophysiologic processes, such as insulin sensitivity, blood pressure, endothelial function, fibrinolytic system, and anti-inflammatory. Adiponectin (ANP) is a kind of protein hormone secreted specifically by adipocyte, which has been reported can improve insulin resistance, against metabolic syndrome, anti-atherosclerosis, and anti-inflammatory. ANP is forward to be the predictor and target for diagnosis and treatment of the obesity related disturbance characterized by insulin resistance, such as metabolic syndrome, type 2 diabetes mellitus and related cardiovascular disease. Retinol binding protein 4 (RBP4) is a new adipocytokine identified recently, which is reported to be involved in the glucoregulation, and would induce insulin resistance. However, the role of RBP4 in the pathogenesis of MS and its regulation factors are still in suspense. The present study aims to identify the role of ANP and RBP4 in metabolic syndrome and the relationship with its components through measuring plasma level of ANP and RBP4. Previous study indicates that there has gender difference in plasma level of ANP and RBP4. The underlying mechanism is partly thought to be related to sex hormones, but is still unknown, thus needs further investigation. In order to explore the underlying mechanism and identify our hypothesis, the present study has evaluated plasma level and expression of ANP and RBP4 in dissected adipose tissue and cultured adipocytes. Project has been divided into 3 parts: (1) Detecting plasma level of APN, RBP4, sex hormones, and sex hormone binding globulin (SHBG) in the patient with different pathophysiologic state of metabolic syndrome, to explore the relationship between these indicators and clinical feature; (2) Examining the expression of APN, Adiponectin receptor 1 (Adipo R1) and RBP4 in intra-abdominal (omental) adipose tissue and subcutaneous tissue, to identify the character of expression for these indicators in central obesity; (3) Effect of sex hormones on differentiation and adipogenesis of 3T3-L1 preadipocyte, and the expression of APN, Adipo R1 and RBP4 in the course of preadipocyte differentiation, to explore the underlying mechanism in gender difference of plasma level of APN and RBP4, and the role of sex hormones.Material and Methods:The project is composed of clinical observation and experimental investigation. General information and plasma characters of patients were detected in the part of clinical observation. Expression of interested proteins was measured in intra-abdominal and subcutaneous adipose tissue, and in the course of 3T3-L1 preadipocyte differentiation.1. Patients were assigned into MS group, non-MS group, and healthy control group. Parameter of body fat (BMI, WC, and WHR) and blood pressure were measured, general blood characters, plasma level of glucose, lipid, inflammatory indicator, APN and RBP4 were detected, and cigarette smoking and alcohol intake were surveyed with subjective questionnaire, in order to identify plasma level of APN and RBP4 in diverse disorders and its regulatory factors.2. Patients were divided into (central) obesity group and non-obesity group. General blood characters, plasma level of APN, RBP4, sex hormones, and SHBG were detected for the investigation of molecular mechanism, especially the role of sex hormones in the gender difference of APN and RBP4 level.3. The presence of APN, Adipo R1 and RBP4 in intra-abdominal and subcutaneous adipose tissue was detected by immunofluorescence. Expression of APN, Adipo R1 and RBP4 was measured with western blotting, in order to clarify the relationship among plasma level, protein expression in adipose tissue, and the plasma level of sex hormones. 4. 3T3-L1 preadipocytes were cultured with estradiol of 10-10M (low level), 10-8M (physiological level), and 10-6M (high level), and testosterone of 3×10-10M (low level), 3×10-8M (physiological level), and 3×10-6M (high level), then the adipogenesis was evaluated with oil red staining.5. 3T3-L1 preadipocytes in state of differentiation inducing, non-differentiation inducing, and maturation were cultured with estradiol of 10-10M (low level), 10-8M (physiological level), and 10-6M (high level), and testosterone of 3×10-10M (low level), 3×10-8M (physiological level), and 3×10-6M (high level), or without sex hormones. Expression of APN, Adipo R1 and RBP4 was measured with western blotting, in order to investigate does-dependent effect on APN, Adipo R1 and RBP4 expression in 3T3-L1 preadipocyte and mature adipocyte. In addition, molecular mechanism for the gender difference of APN and RBP4 will be identified in this sectionResults:1. Results of clinical observation: (1) Plasma level of RBP4 in MS group is significant higher than control group, while APN in MS group is decreased significantly. (2) Plasma level of RBP4 in male is significant higher, and APN is lower than that in female. (3) Plasma level of RBP4 is relation to gender and BMI independently, while plasma level of APN is relation to WC and level of RBP4.2. Results of clinical observation: (1) Plasma level of RBP4 in obesity group (BMI≥25) is significant higher than control group, while APN in obesity group (BMI≥25) group is decreased significantly. (2) Subgroup analysis for male and female, the results (1) also establish. (3) Plasma level of SHBG in obesity group (BMI≥25) decreases significantly. (4) In male, plasma level of RBP4 is independently relation to BMI, WHR, and level of APN; plasma level of APN is independently relation to WC, HDL-C, age and level of RBP4. In female, plasma level of RBP4 is independently relation to BMI and level of LH; plasma level of APN is independently relation to WC and level of hsCRP.3. The presence of APN, Adipo R1 and RBP4 in intra-abdominal and subcutaneous adipose tissue was determined by immunofluorescence.4. Results of western blotting: (1) Expression of APN is decreasing significantly in intra-abdominal adipose tissue of MS patients. Expression of APN in intra-abdominal adipose tissue of male MS subjects is significant lower than female, while in subcutaneous adipose tissue of female MS subjects is significant higher than male. In non-MS group, expression of APN in both kind of adipose tissue is similar. (2) Expression of Adipo R1 in intra-abdominal adipose tissue of female MS subjects is significant lower than control group, but is higher than that in male. Expression of Adipo R1 in intra-abdominal and subcutaneous adipose tissue of female MS subjects is significant higher than that in male. Expression of Adipo R1 in subcutaneous adipose tissue of male MS subjects is significant higher than that in intra-abdominal adipose tissue. (3) Expression of RBP4 in intra-abdominal adipose tissue from male MS patients is decreasing significantly than control group or female patients. Expression of RBP4 in subcutaneous adipose tissue of male subjects is significant lower than female subjects. Expression of RBP4 in intra-abdominal adipose tissue of female subjects is increasing significantly. However, this difference was not been observed in male subjects.5. Comparison of lipogenesis of 3T3-L1 preadipocyte with oil red staining: (1) in non-differentiation inducing group, lipogenesis after intervention with estradiol only in the concentration of 10-8 M for 72 hours is increasing significantly than control group. (2) In differentiation inducing group, lipogenesis after intervention with sex hormone is similar with control group. (3) In the mature adipocyte, lipogenesis after intervention with estradiol in the concentration of 10-10 M or 10-8 M, and testosterone in the concentration of 3×10-8 M is increasing significantly than control group, while that with testosterone in the concentration of 3×10-10 M is decreasing significantly.6. Culture of 3T3-L1 preadipocyte: (1) in non-differentiation inducing group, expression of APN after intervention with estradiol only in the concentration of 10-10 M, and in any concentration of testosterone is increasing significantly than control group;Expression of Adipo R1 after intervention with testosterone in the concentration of 3×10-10 M and 3×10-6 M is increasing significantly than control group; Expression of RBP4 after intervention with testosterone in the concentration of 3×10-10 M is significant lower than control group. (2) In differentiation inducing group, expression of APN after intervention with estradiol testosterone in the concentration of 3×10-8 M is increasing significantly than control group; Expression of Adipo R1 after intervention with testosterone in the concentration of 3×10-8 M and 3×10-6 M is increasing significantly, while that with 10-6 M estradiol is significant lower; Expression of RBP4 after intervention with testosterone in the concentration of 3×10-8 M and 3×10-6 M is increasing significantly, while that with 10-6 M estradiol is significant lower. (3) In the mature adipocyte, expression of APN after intervention with estradiol in the concentration of 10-8 M and testosterone in the concentration of 3×10-8 M is increasing significantly; Expression of Adipo R1 after intervention with any level of testosterone is significant higher; Expression of RBP4 after intervention with estradiol in the concentration of 10-10 M and10-8 M, or any level of testosterone is significant higher than control group.Conclusion:1. Plasma level of RBP4 in MS patient is significant increasing, while APN is significnat lower than control group. Plasma level of RBP4 and APN is closed to components of MS, moreover plasma level of RBP4 and APN is related independently. In additon, RBP4 and APN would become the indicators of obesity and obesity related disorders.2. Disorders of sex hormone level in MS patients are change to reverse trands with gender difference, and level of SHBG is significant decreasing. Plasma level of RBP4 and APN changes significantly to the gender difference, and is closely related to the level of sex hormones and SHBG.3. Expression of APN, Adipo R1 and RBP4 is present in human intra-abdominal and subcutaneous adipose tissue. (1) Expression characters of APN in intra-abdominal adipose tissue is similar to plasma level of APN, which indicates that lower level of APN in patients with central obesity or MS is attributed to the significant lower secration of APN from intra-abdominal adipose tissue. (2) Expression characters of Adipo R1 in intra-abdominal adipose tissue is similar to plasma level of APN, which indicates that level of APN can enhance the biological effect of APN through positive regulation on its expression level. (3) Expression characters of RBP4 in intra-abdominal adipose tissue is different to plasma level, which indicates that increasing plasma level in central obesity, MS patients, and male patients is not from intra-abdominal adipose tissue, but related to the metabolic and functional state of metabolic organs.4. Lipogenesis of preadipocyte is regulated by estradiol and testosterone. Physiologic or lower level of estradiol and physiologic level of testosterone can induce, but lower level of testosterone will suppress lypogenesis in mature adipocyte. 5. Expression of APN, Adipo R1, and RBP4 in preadipocyte and adipocyte is regulated by sex hormones. Plasma level of SHBG is decreasing in patients with MS, and testosterone level in female MS patients is higher than non-MS group. In mature adipocyte, expression of APN is induced by physiologic level of estradiol and testosterone. Expression of Adipo R1 is induced by testosterone, especially in physiologic level. Expression of RBP4 in patients with central obesity and MS is not anly regulated by sex hormones, but also by other factors, such as metabolic state. Higher RBP4 and lower ANP level maybe a protective mechanism in MS.更多还原