【作者】 张苏河；

【导师】 陈璐璐；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 研究背景与目的妊娠期糖尿病(gestational diabetes mellitus,GDM)是妊娠期发生或首次识别出的不同程度的葡萄糖耐量异常,严重危及母婴健康并与多种不良妊娠结局密切相关。GDM是2型糖尿病(type 2 diabetes,T2DM)的高危因素,尽早诊断并治疗GDM不仅可减少围产期母婴死亡率,并可能减少孕妇将来T2DM的发生。GDM的发病机制与胰岛素抵抗(insulin resistance,IR)和胰岛功能减退有关。严重胰岛素抵抗的发生提示GDM患者与T2DM有共同发病机制。近年来参与其中的脂肪细胞因子及炎症因子成为新的研究热点。脂联素是脂肪细胞分泌的一种细胞因子,具有抗炎、抗糖尿病、抗动脉粥样硬化和增强胰岛素敏感性的作用。T2DM患者脂联素水平降低。以往脂联素与胰岛素抵抗机制关系的研究多集中于T2DM患者,对GDM的研究甚少。研究还显示T2DM患者血中游离脂肪酸(free fatty acid,FFA)升高,是一个独立的危险因素,而升高的FFA可导致胰岛素抵抗和胰岛β细胞的凋亡。此外,对T2DM的病因与发病机制的研究中,近年来炎症学说备受关注,低度炎症反应在T2DM的发病机制中扮演着重要角色。炎症因子C反应蛋白(C reactive protein,CRP)已被证实是T2DM事件的预测因子。作为T2DM高危因素的GDM,GDM患者血脂联素与FFA、CRP、IR关系如何,其网膜和皮下脂肪组织中的脂联素的表达如何,骨骼肌脂联素受体1有何变化,他们与IR有关因素的相关性如何,是否与2型糖尿病有类似的改变?这些问题鲜见研究。本研究以GDM为研究对象,设正常妊娠及非妊娠正常血糖组为对照1)观察脂联素、CRP及FFA等在GDM患者血中的变化,探讨其与IR及GDM发病的关系。2)用RT-PCR方法检测腹部皮下脂肪和大网膜脂肪脂联素mRNA、腹直肌脂联素受体1mRNA的表达水平,并比较它们与多种指标的关系,探讨GDM脂肪组织中的脂联素mRNA及腹直肌脂联素受体1mRNA表达水平变化与GDM IR和发病机制的关系。材料与方法受试对象经75克葡萄糖耐量试验后分为3组:①妊娠期糖尿病组(GDM组)②正常妊娠组(normal glucose tolerance,NGT)③非妊娠正常血糖对照组(normal control,NC)。方法(1)用ELISA法检测妊娠38周—40周GDM组、NGT、NC组血脂联素,同时检测FFA和CRP、空腹胰岛素、C肽,总胆固醇(total cholesterol,TC)、甘油三脂(triglyceride,TG)、高密度脂蛋白(high-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白(low density lipoprotein cholesterol,LDL-C),测量身高、体重,计算体质指数(BMI)=体重(kg)/身高~2(m~2),采用稳态模型法(Homeostasis modelassessment,HOMA)评价胰岛素抵抗指数和胰岛β细胞功能即HOMA-IR=FINS×FPG/22.5;HOMA-β=ln[20×FINS/(FPG-3.5)]。通过统计分析,揭示血脂联素与各项指标之间的相互关系。(2)GDM组、NGT组剖腹产时、非妊娠正常血糖对照组(NC)手术时,患者知情同意的情况下在手术过程中取腹部皮下脂肪、大网膜脂肪及腹直肌组织标本,用于RNA提取。RT-PCR方法检测皮下脂肪和大网膜脂肪脂联素mRNA、腹直肌脂联素受体1 mRNA的表达水平。结果(1)各组血中测定指标比较①从NC组、NGT组到GDM组血脂联素水平逐渐降低,各组间差异有显著性(均P＜0.05);FFA水平逐渐升高,各组间差异有显著性;CRP逐渐升高,GDM组与NGT组和NC组间差异有显著性,而NGT组和NC组差异没有显著性。②GDM组血脂联素水平与HOMA-IR呈负相关关系(r=-0.560,P＜0.01),FFA与HOMA-IR呈正相关关系(r=0.515,P＜0.01),CRP水平与HOMA-IR呈正相关关系(r=0.640,P＜0.01)。③GDM组中以HOMA-IR为因变量的多元逐步回归分析显示,CRP、孕前BMI和脂联素最后进入方程,成为影响HOMA-IR最显著的因素。以脂联素为因变量的逐步多元回归分析显示,CRP和孕前BMI最后进入方程,成为影响脂联素最显著的因素。(2)剖腹产(GDM组、NGT组)及非孕手术对照组(NC组)①各组皮下、网膜脂肪组织脂联素mRNA表达量、各组腹直肌组织脂联素受体1 mRNA表达量从NC组、NGT组到GDM组逐渐降低,各组间差异有显著性(P＜0.01,P＜0.05);网膜脂肪组织脂联素mRNA表达量下降较皮下脂肪组织更明显。②GDM组中皮下脂肪组织脂联素mRNA表达量与HOMA-β、LDL-C呈正相关;网膜脂肪组织脂联素mRNA表达量与FPG、FINS、HOMA-IR呈负相关性,与HOMA-β呈正相关性;腹直肌组织脂联素受体1mRNA表达量与FPG、HOMA-IR、TC、TG负相关。结论(1)GDM患者存在严重的IR,;血脂联素、CRP和孕前BMI与IR独立相关;胰岛素抵抗参与GDM的发生。(2)相对于正常妊娠组,GDM组CRP水平显著升高,FFA水平显著升高,提示慢性炎症反应和脂代谢紊乱与严重IR及GDM的发病有关。GDM组的血脂联素水平显著降低,且与HOMA-IR负相关,提示脂联素降低与严重IR及GDM的发病有关。(3)GDM患者腹壁皮下、网膜脂肪组织脂联素mRNA水平均明显低于健康孕妇组及正常对照组;网膜脂肪组织脂联素mRNA水平下降更明显,提示其改变与GDM发生有关。(4)GDM组网膜脂肪组织脂联素mRNA表达量与FPG、FINS、HOMA-IR呈负相关,网膜脂肪组织脂联素mRNA表达量与胰岛素抵抗有关,GDM呈现与2型糖尿病一致的改变。(5)GDM组骨骼肌组织脂联素受体1 mRNA表达量低于健康孕妇组及正常对照组,各组间差异有显著性(P＜0.05)。GDM脂联素受体1mRNA表达量下降与糖脂代谢异常、IR密切相关。GDM血脂联素、皮下、网膜脂肪组织脂联素mRNA表达量下降,同时骨骼肌脂联素R1基因表达下调,进一步导致了脂联素不能有效结合受体,形成恶性循环。更多还原

【Abstract】 Backgrouds and objectivesGestational diabetes mellitus(GDM),a form of glucose intolerance which occurred during pregnancy,could do great harm to the health of pregnant women and fetus,and has been known for being closely correlated with poor outcomes of gestation.GDM is a risk factor for the later development of type 2 diabetes,and timely intervention to GDM may not only significantly decrease the mortality rate of fetus during perinatal period,but delay the onset of type 2 diabetes in women.Both insulin resistance and beta cell dysfunction have been thought being involved into the pathogenesis of GDM,while chronic insulin resistance share common mechanism in GDM and type 2 diabetes.Previous studies have found that C-reactive protein(CRP) and IL-6 are the predictors for type 2 diabetes, however whether cytokines could predict the onset of GDM or play an critical role in the progression of GDM still is unknown.Free fatty acids(FFA) is the critical factor in the metabolism of lipid.It has been reported that elevated level of plasma FFA in type 2 diabetes subjects is an independent risk factor,and could lead to insulin resistance and increased apoptosis rate of beta cells,which may be conducted by the inflammation.Adiopnectin,one of the most important adipokines, is believed to possess the bioactivity like anti-inflammation,anti-diabetes,anti-artherosclerosis and improve insulin sensitivity.Not surprisingly,the level of plasma adiponectin was decreased in the type 2 diabetes patients.The studies concerning the association between GDM and adiponectin are few,though many have been done in the fields of its involvement in type 2 diabetes.In the present study,80 subjects were stratified into three groups according to the fasting and 2h blood glucose:GDM group,normal glucose tolerance with gestation group and normal glucose.tolerance without gestation group.The expression level of adiponectin and its receptor in subcutaneous/abdominal fat and muscle was compared in these groups, so did other cytokines and FFA.Furthermore,the association between adiponectin and its receptor and insulin resistance in GDM was also assessed in this study.The measurements of serum CRP,adiponectin and FFA were then performed in these subjects,and the expression level of adiponectin and its receptor was detected in subcutaneous fat, abdominal fat and muscle tissues from these groups by using RT-PCR.Subjects and methodsBased on the results of 75g oral glucose tolerance test,90 subjects were stratified into three groups according to the fasting and 2h blood glucose:GDM group,normal glucose tolerance with gestation group and normal glucose tolerance without gestation group. Anthropometric measurements included:height,weight,body mass index(BMI,kg/m~2). Blood samples for measurements of free fatty acid(FFA) and C peptide,total cholesterol (TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol (HDL-C),triglyceride(TG) and CRP levels were collected after an overnight fast.Plasma glucose was assayed by an automated glucose oxidase method.Insulin was determined by RIA.The concentration of serum adiponectin was measured by ELISA method.The homeostasis model(HOMA) model was adopted to calculate the index of insulin resistance HOMA-IR=FINS×FPG/22.5) and beta cell function(HOMA-β=20×FINS/(FPG-3.5)).The expression level of adiponectin and its receptor was detected in subcutaneous fat, abdominal fat and muscle tissues from these groups by using RT-PCR.The study was designed in compliance with the ethics regulations set out by the Helsinki Declaration,and all subjects were well informed about the study before the consent was acquired.Results1.During the period between 38-40 weeks after gestation,①The level of adiponectin was gradually decreased from NC group,NGT group to GDM group,and the difference between among these groups was significant(P＜0.05);The level of FFA was gradually increased from NC group,NGT group to GDM group,and the difference among these groups also was significant(P＜0.05);The level of CRP in GDM group was higher than those of NGT and NC group respectively(P＜0.05).②In GDM group,the plasma level of adiponectin was negatively correlated with HOMA-IR(r=-0.560,P＜0.01),while the plasma level of FFA and CRP was positively correlated with HOMA-IR respectively(r=0.515,P＜0.01;r=0.640,P＜0.01);③Stepwise regression analysis indicated that CRP,BMI(before gestation) and adiponectin was the last ones enter the equations,when HOMA-IR was taken as dependent variable.While in the stepwise regression analysis,in which adiponectin was taken as dependent variable,CRP and weight entered the equations last.2.During the period from38-40 weeks after gestation,①The mRNA expression of adiponectin and its receptor in subcutaneous and abdominal fat was gradually decreased from NC group to GDM group(P＜0.05).②In GDM group,the mRNA expression of adiponectin in subcutaneous adipose tissue was positively correlated with HOMA-β,and the level of LDL-C;the mRNA expression of adiponectin in abdominal adipose tissue was negatively correlated with FPG, FINS and HOMA-IR;the mRNA expression of adiponectin receptor was negatively correlated with FPG,HOMA-IR,TC and TG.Conclusions(1) GDM subjects has increased insulin resistance,and CRP,BMI(before gestation) and plasma adiponectin was independently associated with IR,which demonstrated that the insulin resistance was involved in the onset of GDM.(2) The level of CRP and FFA was higher in GDM than that of gestation group with normal glucose tolerance,which suggested the chronic inflammation was associated with lipidemia and insulin resistance.The level of plasma adiponectin was significantly decreased in GDM,and negatively correlated with HOMA-IR,which indicated that the adiponectin played an role in the insulin resistance and the onset of GDM.(3) The level of mRNA expression of adiponectin in subcutaneous adipose tissue was significantly decreased in GDM subjects than those of gestation group and normal control, while the abdominal tissue decreased even more,indicating its role in GDM.(4) The mRNA expression of adiponectin in abdominal adipose tissue in GDM was negatively correlated with FPG,FINS and HOMA-IR,which suggest that the adiponectin in abdominal adipose tissue is involved in the development of insulin resistance.(5)The level of mRNA expression of adiponectin receptor 1 in muscle tissue was lower than that in normal gestation group and control group.(P＜0.05),which means this decrease was closely correlated with glucolipid metabolism and insulin resistance.更多还原