【作者】 张丹；

【导师】 张华；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2023， 硕士

【摘要】 目的:妊娠期糖尿病(gestational diabetes mellitus,GDM)主要特征为餐后血糖调节异常,目前空腹血糖测定对GDM的诊断价值已经十分有限。近年来,中国人的饮食结构发生巨大改变,GDM的发病率逐年增高,已经高达17.5%。餐后血糖除受胰岛素及胰高血糖素调控外,同时也受胃肠胰岛轴的动态调节。妊娠期特有的胃肠功能失调可能推动GDM发生。大量研究指出,胃肠胰岛素轴中胆汁酸的肠肝循环在调节血糖方面发挥重要的作用。胆汁酸与雌孕激素具有相似的化学结构,可能会在肝脏代谢过程中发挥交叉作用。此外,大鼠雌激素颈部皮下注射法已经是妊娠期胆汁淤积症的成熟动物模型。有研究指出,发生妊娠期肝内胆汁淤积的孕妇同时发生妊娠期糖尿病的几率更大。这从侧面证明了胆汁酸代谢情况与血糖的调节机制密切相关。所以本研究主要关注雌孕激素对于胆汁酸受体、胆汁酸转运体的影响,这种影响进而导致部分孕妇出现胆汁酸转运障碍,排入小肠的胆汁酸减少,进而GLP-1生成减少,使餐后血糖异常升高,导致GDM。方法:(1)临床研究中,收集100例妊娠期糖尿病妇女和100例健康妇女的24-28w 75g OGTT试验时的血浆样本作为正常对照。采用液相色谱-质谱(liquid chromatography-mass spectrometry,LC-MS)分析对照组和GDM组的血浆胆汁酸和性激素代谢物的差异。采用Elisa法检测对照组和GDM组血浆中GLP-1、空腹胰岛素的水平。采用罗氏cobas8000 c701检测对照组和GDM组空腹血浆的脂质蛋白的差异。(2)用正常饮食+溶剂、高脂饮食+溶剂、正常饮食+雌二醇、正常饮食+孕酮颈部皮下注射法构建大小鼠动物模型,用LC-MS检测动物模型中肝脏,各部分肠道组织及其内容物的胆汁酸水平的差异。用WB和q PCR检测动物样本中肝脏的关键的胆汁酸转运体FXR、CYP7A1、NTCP、BSEP蛋白和m RNA的表达情况。(3)从雌性小鼠中提取原代肝细胞做“三明治”肝细胞培养。结果:(1)GDM孕妇和对照组相比,餐后胆汁酸调节的模式发生变化,在GDM组胆汁酸和性激素代谢受到影响,用Mann-Whitney秩和检验时,我们发现GDM组和对照组的空腹血浆胆汁酸没有差异。但是餐后1h和餐后2h后GDM组血浆总胆汁酸出现降低。就结合型胆汁酸来看,相较于对照组,GDM组在餐后2h时血浆胆汁酸水平出现锐减。用双因素方差分析时我们发现在餐后调节血糖的胆汁酸中,GDM与对照组出现明显差异的是非结合型胆汁酸石胆酸(LCA)和鹅脱氧胆酸(CDCA)。(2)在雌孕激素代谢产物的分析中,用双因素方差分析检验时我们发现在GDM组中空腹的雌孕激素代谢物升高,特别是雄烯二酮,肾上腺甾酮水平升高。(3)动物实验表明雌激素注射会导致动物的糖代谢异常。WB和q PCR结果显示肝脏转运体FXR、CYP7A1、NTCP和m RNA的表达下调。(4)细胞层面我们主要建立了“三明治肝细胞”模型。结论:GDM组胆汁酸代谢异常,血浆中总循环胆汁酸水平和结合型胆汁酸水平较低,雌孕激素代谢物会影响胆汁酸的代谢,这可能是GDM餐后血糖调节机制受损的原因之一。更多还原

【Abstract】 Objective: The main characteristic of gestational diabetes mellitus(GDM)is abnormal postprandial blood glucose regulation.The diagnostic value of fasting blood glucose measurement for GDM is very limited.In recent years,the diet structure of Chinese people has changed greatly,and the incidence of GDM has increased year by year,reaching 17.5%.Postprandial blood glucose is not only regulated by insulin and glucagon,but also by the dynamic regulation of gastrointestinal islet axis.Gastrointestinal disorders specific to pregnancy may promote the development of GDM.A large number of studies have pointed out that the enterohepatic circulation of bile acids in the gastrointestinal insulin axis plays an important role in regulating blood sugar.Bile acids have a similar chemical structure to estrin and may play a cross-over role in liver metabolism.In addition,subcutaneous injection of estrogen in the neck of rats has been a well-established animal model of gestational cholestasis.It has been suggested that pregnant women with intrahepatic cholestasis of pregnancy have an increased risk of developing gestational diabetes.This proves from the side that bile acid metabolism is closely related to the mechanism of blood glucose regulation.Therefore,this study mainly focused on the influence of estrogen on bile acid receptors and bile acid transporters,which in turn led to the obstruction of bile acid transport in some pregnant women and the decrease of bile acid drained into the small intestine,thus reducing the production of GLP-1,resulting in the abnormal rise of postprandial blood sugar and leading to GDM.Methods:(1)In the clinical study,plasma samples of 100 women with gestational diabetes mellitus and 100 healthy women during the 24-28 W 75g OGTT test were collected as normal controls.liquid chromatography-mass spectrometry(LC-MS)was used to analyze the differences of plasma bile acid and sex hormone metabolites between the control group and the GDM group.Plasma levels of GLP-1 and fasting insulin in control group and GDM group were determined by Elisa.The difference of fasting plasma lipid protein between the control group and the GDM group was determined by Roche cobas8000 c701.(2)Animal models of rats and mice were constructed by subcutaneous injection of normal diet+ solvent,high-fat diet + solvent,normal diet + estradiol,normal diet +progesterone in the neck.The bile acid levels of liver,intestinal tissues and their contents in the animal models were detected by LC-MS.The protein and m RNA expression of key bile acid transporters FXR,CYP7A1,NTCP and BSEP in liver were detected by WB and q PCR.(3)Primary hepatocytes were extracted from female mice for "sandwich" hepatocyte culture.Results:(1)Compared with the control group,postprandial bile acid regulation pattern was changed in pregnant women with GDM.Bile acid and sex hormone metabolism were affected in the GDM group.When Mann-Whitney rank sum test was used,we found no difference in fasting plasma bile acid between the GDM group and the control group.However,plasma total bile acids decreased in GDM group 1h and 2h postprandial.In terms of binding bile acids,compared with the control group,plasma bile acids in GDM group decreased sharply 2h after meal.In the two-way analysis of variance,we found that among the bile acids that regulate blood glucose after meal,the unbound bile acids,stone cholic acid(LCA)and goose deoxycholic acid(CDCA),showed significant differences between GDM and the control group.(2)In the analysis of estrogen metabolites,it was found that fasting estrogen metabolites,especially androstenedione and adrenosterone levels,were increased in the GDM group when tested by two-way analysis of variance.(3)Animal experiments have shown that estrogen injection can cause abnormal sugar metabolism in animals.The expressions of FXR,CYP7A1,NTCP and m RNA in liver were downregulated by WB and q PCR.(4)At the cellular level,we mainly established the "sandwich hepatocyte" model.Conclusions: Bile acid metabolism is abnormal in GDM group,and the level of total circulating bile acid and binding bile acid in plasma is lower.Estrogen and progesterone metabolites can affect the metabolism of bile acid,which may be one of the reasons for the impaired mechanism of postpranpranal blood glucose regulation in GDM group.更多还原