【作者】 马蓉；

【导师】 刘建； 肖晓秋；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2011， 硕士

【摘要】 目的:本研究旨在探讨妊娠期糖尿病(gestational diabetes mellitus,GDM)胎盘组织I型和II型11β羟基类固醇脱氢酶(11β-HSD1和11β-HSD2)基因和蛋白表达的变化,分析11β-HSDs的差异表达与母婴体内糖皮质激素水平之间的关系,进而揭示妊娠环境中皮质醇的变化在GDM发病与胎儿生长发育过程中的意义。方法:1.选择重庆医科大学附属第二医院同期分娩的妊娠期糖尿病(GDM组)23例和糖耐量正常孕妇(NGT组)22例,分离收集两组产妇手术当天的肘静脉血、脐血和胎盘组织。2.全自动生化分析仪检测母血的各项生化指标,葡萄糖氧化酶法测定手术当天空腹血糖水平,放射免疫法测定空腹胰岛素水平,化学发光法检测皮质醇的含量,稳态模型评估法(HOMA)计算胰岛素抵抗指数(HOMA-IR)和胰岛素分泌指数。3.免疫组织化学法检测胎盘组织中11β-HSD1和11β-HSD2表达部位,Real time-PCR和Western blot法检测NGT组和GDM组中11β-HSD1和11β-HSD2 mRNA和蛋白水平的表达。4.取糖耐量正常孕妇的足月胎盘绒毛组织,给予游离脂肪酸、地塞米松、胰岛素及含以上几种物质的混合物体外孵育24h,Real time-PCR和Western blot法检测组织孵育后11β-HSD1和11β-HSD2 mRNA和蛋白水平的表达,以确定在妊娠期糖尿病条件下,代谢和体液因素调节胎盘组织11β-HSDs的分子机制。结果: 1.与NGT组相比,GDM组空腹胰岛素(14.8±1.5和19.8±1.7(μU/ml))、HOMA-IR(2.8±0.3和4.1±0.4)、胰岛素分泌指数(57.2±5.8和83.7±7.3)、母血皮质醇水平(940.3±44.2和1125.0±42.8(nmol/L))、甘油三酯(3.3±0.4和4.3±0.4(nmol/L))和产时BMI(26.8±0.8和29.0±0.6 (kg/m2))显著增高(P<0.05),而空腹血糖、脐血皮质醇、新生儿体重、高密度脂蛋白、低密度脂蛋白则无明显差异。2.合并两组静脉血皮质醇和脐血皮质醇结果,新生儿体重与母亲静脉血皮质醇无明显相关性,与脐血中皮质醇水平呈明显负相关(R=-0.378,P=0.01)。3.11β-HSD1蛋白在胎盘散在分布于绒毛单位间质,绒毛干的合体滋养层,绒毛合体滋养层外层;11β-HSD2蛋白表达集中分布于绒毛合体滋养层外层。4. Real time-PCR结果提示GDM组胎盘11β-HSD1 mRNA的表达明显低于NGT组,分别为(0.56±0.09和1.36±0.36,P<0.05);11β-HSD2 mRNA明显高于NGT组(5.17±1.02和1.21±0.34,P<0.05)。Western blot法结果提示GDM组胎盘11β-HSD1蛋白水平的表达明显低于NGT组,分别为(0.27±0.07和1.00±0.19,P<0.05),而11β-HSD2蛋白水平增加无统计学意义(1.30±0.19和1.00±0.14,P>0.05)。5.取糖耐量正常孕妇的足月胎盘绒毛组织,给予游离脂肪酸、地塞米松、胰岛素及含以上几种物质的混合物体外孵育和刺激24h,Real time-PCR结果提示不同处理组中11β-HSD1 mRNA的表达明显低于基础状态未处理组,未处理组、游离脂肪酸组、地塞米松组、胰岛素组、混合物处理组分别为(1.00±0.01;0.49±0.04;0.57±0.16;0.44±0.07;0.55±0.13,P<0.05),11β-HSD2 mRNA明显高于基础状态未处理组,(各组分别为1.00±0.01;1.14±0.12;1.43±0.12;1.47±0.13;1.91±0.17,P<0.05)。Western blot法提示给予体外孵育和刺激组织后,不同处理组中11β-HSD1蛋白水平表达低于基础状态未处理组,(各组分别为0.99±0.01;0.36±0.03;0.13±0.01;0.68±0.09;0.50±0.04, P<0.05),11β-HSD2蛋白水平表达高于基础状态未处理组,(各组分别为1.04±0.04;2.35±0.18;4.10±0.98;3.20±0.39; 3.91±0.32, P<0.05)。结论:(1) GDM患者经过饮食、运动控制后空腹血糖恢复,但仍存在明显胰岛素抵抗。与NGT组相比,GDM患者存在高胰岛素血症、高脂血症、高皮质醇血症。(2) GDM患者血清中皮质醇浓度明显升高,然而胎儿脐血中皮质醇与胎儿体重均无明显变化。胎儿体重与母血中皮质醇无明显相关性,与脐血中皮质醇呈负相关。(3)妊娠足月胎盘中存在11β-HSD1和11β-HSD2表达,GDM患者胎盘11β-HSD1表达的下调和11β-HSD2表达的上调共同参与了脐血中皮质醇水平的稳态维持,循环中的高皮质醇、高脂血症、高胰岛素是引起胎盘11β-HSDs变化的原因。(4)胎盘组织11β-HSDs的表达变化提供了一种对胎儿的保护机制,调节糖皮质激素自母体经胎盘向胎儿体内的转运,GDM发生时,11β-HSDs调节机制避免母体不利的妊娠环境对胎儿可能产生的深远损害。更多还原

【Abstract】 Objective: Our current studies were designed to investigate placental type 1 and 2 of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) gene and protein expression among pregnant women either with gestational diabetes mellitus (GDM) or normal glucose tolerance (NGT). In addition, correlation between fetal growth and maternal and fetal cortisol levels with placental 11β-HSDs was analyzed.Methods: 1. Age matched pregnant women with gestational diabetes mellitus (GDM group, n=23) and normal glucose tolerance (NGT group, n=22) were recruited at the 2nd Affiliated Hospital, CQMU. The blood from maternal and umbilical venous was collected and placental tissues were dissected. 2. The biochemical index and fasting glucose of maternal blood were analyzed by automatic biochemical analyzer and glucose oxidase. Cortisol and insulin levels were examined by chemiluminescence and radioimmunoassay (RIA). The homeostasis model assessment of insulin resistance index (HOMA-IR) and insulin secretion index were performed as literatures. 3. Immunohistochemical assay were applied for measurement of 11β-HSD1 and 11β-HSD2 distribution in the placenta. Real time-PCR and Western blot were applied for measurement of 11β-HSD1 and 11β-HSD2 mRNA and protein expression between NGT group and the GDM group. 4. Placental villi explants from full-term NGT pregnancy were incubated with free fatty acids , dexamethasone, insulin and their combined mixture for 24h. Real time-PCR and Western blot were used to measure 11β-HSD1 and 11β-HSD2 mRNA and protein expression in incubated placental explants.Results: 1. Compared with NGT group, fasting insulin (14.8±1.5 vs 19.8±1.7 (μU / ml)), HOMA-IR (2.8±0.3 vs 4.1±0.4), insulin secretion index (57.2±5.8 vs 83.7±7.3), maternal cortisol levels (940.3±44.2 vs 1125.0±42.8 (nmol / L)), triglycerides (3.3±0.4 vs 4.3±0.4 (nmol / L)) and BMI (26.8±0.8 vs 29.0±0.6 ( kg/m2)) from GDM group were significantly higher, respectively, P <0.05), while fasting blood glucose, cord blood cortisol ,birth weight, high density lipoprotein and low density lipoprotein did not differ between the two groups. 2. Pearson’s correlation analysis between maternal vein or umbilical cord cortisol levels and fetal body weight demonstrated a negative correlation between fetal body weight with umbilical cord cortisol levels (R=-0.378, P=0.01) but not maternal vein cortisol levels. 3. 11β-HSD1 protein in the placental villi was extensively distributed in the villous unit interstitial substance, syncytiotrophoblast of villious branches, and outer syncytiotrophoblast, while 11β-HSD2 was relatively concentrated on the outer layer of syncytial trophoblast. 4. Real time-PCR and Western blot results suggest that GDM placental 11β-HSD1 mRNA and protein levels were significantly lower than the NGT group (0.56±0.09 vs 1.36±0.36 for mRNA and 0.27±0.07 vs 1.00±0.01 for protein, respectively, P <0.05). 11β-HSD2 mRNA was significantly higher than NGT group (5.17±1.02 vs 1.21±0.34, P <0.05), while no significant difference was found for 11β-HSD2 protein (1.30±0.19 vs 1.00±0.14, P> 0.05). 5. Treatment of placenta explants from NGT with palmitic acid, dexamethasone, insulin or their combination resulted in a significant drop of 11β-HSD1 and increase in 11β-HSD2. 11β-HSD1 mRNA levels in untreated, palmitic acid, dexamethasone, insulin and combined mi xture explants were : (1.00±0.01, 0.49±0.04, 0.57±0.16, 0.44±0.07, and 0.55±0.13, respectively ,P <0.05); and 11β-HSD1 protein levels were (0.99±0.01, 0.36±0.03, 0.13±0.01, 0.68±0.09, and 0.50±0.04, respectively,P <0.05). 11β-HSD2 mRNA levels in above groups were ( 1.00±0.01, 1.14±0.12, 1.43±0.12, 1.47±0.13, and 1.91±0.17, respectively, P <0.05)and protein levels were (1.04±0.04, 2.35±0.18, 4.10±0.98, 3.20±0.39, and 3.91±0.32, respectively,P <0.05)Conclusions: 1. The fasting blood glucose in GDM patients can be maintained normal through diet control and exercise, even though there are still significant insulin resistance, hyperlipidemia and hypercorticism. 2. Although GDM mothers had significant hypercorticism, fetal cord blood cortisol levels and infant body weight did not significantly changed. Birth weight was not correlated with maternal cortisols, but was negatively correlated with cord blood cortisol levels. 3. Both 11β-HSD1 and 11β-HSD2 were present in the full-term pregnancy placenta. The downregulation of 11β-HSD1 and upregulation of 11β-HSD2 participate in the maintenance of cord blood cortisol levels. High levels of circulating insulin, free fatty acids, and cortisol in GDM state are directly associated with the upregulation of 11β-HSD1 and downregulation of 11β-HSD2 in the placenta. 4. Differential expression of 11β-HSDs in GDM placenta provides a protective mechanism for the fetus throughout the adverse environment of pregnancy by limiting excessive exposure of the fetus to glucocorticoids.更多还原