产前和产后营养过剩对雌性子代小鼠糖脂代谢的影响及机制研究

发布时间：2018-05-17 11:23

  本文选题：营养过剩 + 高脂饮食　； 参考：《山东大学》2017年博士论文

【摘要】：背景:传统观念认为,成人慢性疾病是在多基因遗传易感性的基础上,由包括不良生活方式在内的环境因素所诱发的。1995年,Barker提出了胎儿编程(Fetal programming)假说,认为生命早期的关键窗口对终身健康有"编程效应",生命起始于胚胎和胎儿宫内阶段,宫内环境是否正常关系到个体生命的成长、远期疾病的发生,甚至子孙后代的健康。2010年Motrenkoe又提出了胚胎源性疾病(Embryonic diseases)这个新概念,认为配子和胚胎的发育异常有可能影响子代的健康状态,配子或胚胎对不利环境因素做出适应性反应,最终可能引发子代成年后的糖尿病、肥胖、非酒精性脂肪肝(Non alcoholic fatty liver disease,NAFLD)和心脑血管疾病等疾病。大量研究证实,产前(妊娠前和妊娠期)和产后(哺乳期和断奶后早期)营养过剩都在子代糖脂代谢异常的发生和发展中起到了重要作用。围妊娠期的饮食和生活习惯是亲代和子代健康的重要决定因素。妊娠前超重和肥胖的妇女,流产、妊娠期糖尿病(Gestational diabetes mellitus,GDM)和子痫前期(Preeclampsia,PE)发病率明显增加,高出生体重发生率明显增加,子代成年后肥胖、Ⅱ型糖尿病(Type 2 diabetes mellitus,T2DM)和心血管疾病发病率也明显增加。妊娠期过多的营养摄入是子代健康或疾病的独立危险因素,妊娠期过多的营养摄入与成年后高血压、葡萄糖耐受性受损、非胰岛素依赖型糖尿病(Non-insulin-dependent diabetes mellitus,NIDDM)或 T2DM、胰岛素抵抗和肥胖的发病风险增加的有关,除此之外,癌症也与妊娠期营养过剩有关,有研'究指出,高出生体重者更易罹患乳腺癌和白血病。有研究证实,哺乳期高脂饮食的小鼠的子代成年后可患高血压、血管内皮细胞功能异常、大动脉形态学改变、肾功异常、胰岛素抵抗、糖尿病和胰腺衰竭等疾病,大脑功能异常也有发生。除此之外,子代断奶后早期营养过剩也在将来代谢综合征发病中起到重要作用。然而综合考虑产前和产后营养过剩对雌性子代糖脂代谢影响的研究较少,也很少有研究综合分析产前和产后营养过剩在雌性子代不良代谢结局中的相互作用关系。因此本课题通过构建产前和产后高脂高果糖(High-fat/fructose diet,HFFD)饮食模型,研究产前和产后营养过剩对雌性亲代小鼠和雌性子代小鼠糖脂代谢的影响及作用机制,探讨产前和产后营养过剩在雌性子代不良结局中的相互作用关系。另外,本课题利用生物信息学方法对两组基因芯片表达谱数据进行荟萃分析,筛选产前高脂饮食和产前正常饮食的雌性子代小鼠肝脏的差异表达基因,从基因及其编码蛋白质两个层面深入分析基因芯片表达谱数据,为进一步研究产前营养过剩对雌性子代小鼠脂代谢潜在影响的发生、发展的分子机制提供理论基础。研究方法:1.未孕雌性亲代小鼠分别常规饲料(Normal chow diet,NC)喂养或HFFD喂养。两组雌性亲代小鼠分别进行体重、脂肪含量、血糖、胰岛素水平、胰岛素耐受实验(Insulin tolerance test,ITT)和葡萄糖耐受实验(Glucose tolerance test,GTT)等检测,并进行统计学分析。2.HFFD组和NC组雌性亲代小鼠与NC喂养的雄性亲代小鼠交配;妊娠期,NC组继续NC喂养,HFFD组继续HFFD喂养;雌性子代小鼠随机分组,分别由NC或HFFD喂养的亲代小鼠随机哺乳,这就形成了 NC/NC组,NC/HFFD组,HFFD/NC组和HFFD/HFFD组(产前饮食/产后饮食)这4个不同的组别;断奶后,NC/NC组继续NC喂养,NC/HFFD组继续HFFD喂养,HFFD/NC组继续NC喂养,HFFD/HFFD组继续HFFD喂养。四组雌性子代小鼠分别进行摄食量、能量代谢、体重、空腹血糖、血浆胰岛素水平、血浆甘油三酯(Triglyceride,TG)水平、血浆游离脂肪酸(Free fatty acid,FFA)水平、血浆瘦素水平、ITT实验、GTT实验、脂肪含量、肝脏重量、白色脂肪组织(White adipose tissue,WAT)病理、肝脏病理、胰腺β细胞形态、葡萄糖刺激的胰岛素释放实验(Glucose-stimulated insulin secretion,GSIS)以及肝脏组织和胰腺组织中与糖脂代谢相关的蛋白和mRNA表达等检测,并进行统计学分析。3.在基因表达综合数据库(Gene expression ominibus,GE0)中检索并下载两组产前高脂饮食和产前正常饮食的雌性子代小鼠肝脏基因表达谱数据集;将数据集导入到BRB-Array Tools中标准化处理、整理、分析,获得产前高脂饮食和产前正常饮食的雌性子代小鼠肝脏差异表达基因;向STRING在线分析软件中导入差异表达基因所编码蛋白,获得差异表达基因所编码蛋白的互作网络数据,再将其导入Cytoscape软件中,计算网络及各个节点的拓扑特性,得到最大连通度的蛋白所对应的关键基因;利用基因注释形象集成数据库(The database for annotation visualization and integrated discovery,DAVID)软件进行基因本体论(Gene ontology,GO)功能注释和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析。结果:1.未孕雌性亲代小鼠:HFFD组体形表现出更为明显的肥胖趋势。HFFD组体重、脂肪含量、血糖和胰岛素水平均明显高于NC组。HFFD组ITT和GTT也表现出明显异常。2.雌性子代小鼠:NC/NC组和HFFD/NC组各项指标基本正常;HFFD/HFFD组和NC/HFFD组表现出明显高体重、高脂肪含量、高血糖和严重胰岛素抵抗;与NC/HFFD组相比较,HFFD/HFFD组表现更为明显。HFFD/HFFD组和NC/HFFD血浆瘦素水平明显较高;HFFD/HFFD组血浆瘦素水平比NC/HFFD组更高。HFFD/HFFD组和NC/HFFD组β细胞质量,大小和胰岛素释放情况明显异常;与NC/HFFD组相比较,HFFD/HFFD组变化更加明显。当胰岛素存在时,NC/NC组和HFFD/NC组丝氨酸/苏氨酸激酶(Serine/threoninekinase,Akt)磷酸化显著增加;在胰岛素刺激下NC/HFFD组Akt磷酸化增加,但显著低于NC/NC组或HFFD/NC组;胰岛素刺激不能诱导HFFD/HFFD组Akt磷酸化增加。HFFD/HFFD组和NC/HFFD组胰岛素、过氧化物酶体增生激活受体丫协同刺激因子(Peroxidase body hyperplasia activated receptor gamma stimulating factor,PGC)-1 α 和葡萄糖-6-磷酸酶(Glucose 6 phosphatase,G-6-Pase)蛋白和 mRNA 表达明显高于 NC/NC 组;HFFD/HFFD组与NC/HFFD组相比,胰岛素、PGC-1α和G-6-Pase蛋白和mRNA表达更高。HFFD/HFFD 组和 NC/HFFD 组葡萄糖转运蛋白(Glucose transport protein,GLUT)-2和葡萄糖激酶(Glucokinase,GCK)蛋白和mRNA表达明显低于NC/NC组;HFFD/HFFD组与NC/HFFD组相比,GLUT-2和GCK蛋白和mRNA表达更低。3.产前高脂饮食和产前正常饮食的雌性子代小鼠肝脏差异表达基因85个,其中上调基因38个,下调基因47个。差异表达基因KEGG通路分析得出,有12个基因参与代谢通路,有4个基因参与Wnt信号通路,有4个基因参与磷酸肌醇代谢通路。本研究发现关键基因4个,分别为低密度脂蛋白受体相关蛋白(Low-density lipoprotein receptor-related protein,LRP6)、Prkd2、Kcnk13和 Notch1。结论:1.妊娠前营养过剩可以成功诱导了亲代小鼠脂肪蓄积、高血糖、高胰岛素血症、胰岛素抵抗和葡萄糖耐量异常。2.产前和产后营养过剩的直接结果就是雌性子代小鼠脂肪蓄积,高血糖,高胰岛素血症,和β细胞功能障碍,可以进一步导致其肥胖和T2DM。3.循环中瘦素水平上升和瘦素抵抗是雌性子代小鼠脂肪蓄积的重要原因。胰腺β细胞功能受累,是胰岛素抵抗和糖耐量异常的重要原因。4.Akt磷酸化减少,Akt功能受到抑制;PGC-1 α、胰岛素和G-6-Pase表达升高,糖异生和糖原分解活化;GLUT-2和GCK表达降低,葡萄糖酵解减少;这些都是雌性子代小鼠糖脂代谢异常的重要原因。5.雌性子代小鼠糖脂代谢障碍主要是主要是由产后营养过剩造成的。单纯产前营养过剩不能直接造成雌性子代小鼠糖脂代谢障碍,但对其糖脂代谢功能有重要的潜在影响。6.LRP6,Prkd2,Kcnk13和Notch1是产前高脂饮食和产前正常饮食的雌性子代小鼠肝脏差异表达基因中的关键基因。LRP6在产前高脂饮食对雌性子代小鼠脂代谢潜在影响中发挥重要作用。
[Abstract]:Background: the traditional idea is that adult chronic disease is based on the genetic susceptibility to polygenes and in.1995 years induced by environmental factors including bad lifestyle. Barker proposed the Fetal programming hypothesis that the key window of life has a "programming effect" on life-long health, and life begins from the embryo and the embryo. In the intrauterine stage of the fetus, whether the intrauterine environment is related to the growth of individual life, the occurrence of long-term diseases, and even the health of the offspring of offspring.2010 Motrenkoe also proposed the new concept of Embryonic diseases, which suggests that the abnormal development of gametes and embryos may affect the health of the offspring, gametes or embryos An adaptive response to adverse environmental factors may eventually lead to diabetes, obesity, nonalcoholic fatty liver disease (Non alcoholic fatty liver disease, NAFLD) and cardiovascular and cerebrovascular diseases. A large number of studies have confirmed that prenatal (pre pregnancy and pregnancy) and postpartum (breast-feeding and early weaning) are all in the subgeneration sugar. The occurrence and development of abnormal lipid metabolism have played an important role. The diet and lifestyle in the peri gestation period are important determinants of the health of the parents and offspring. Women who are overweight and obese before pregnancy, abortion, gestational diabetes (Gestational diabetes mellitus, GDM) and preeclampsia (Preeclampsia, PE) have a significant increase in incidence and high birth rate. The incidence of body weight increased significantly, the offspring were obese after adult, type II diabetes (Type 2 diabetes mellitus, T2DM) and cardiovascular disease incidence increased significantly. Excessive nutrition intake during pregnancy was an independent risk factor for offspring health or disease. Excessive nutrition intake during pregnancy and adult hypertension, impaired glucose tolerance, non pancreas "Non-insulin-dependent diabetes mellitus, NIDDM) or T2DM is associated with increased risk of insulin resistance and obesity. In addition, cancer is also associated with excess nutrition in pregnancy. The offspring of mice can suffer from hypertension, abnormal function of vascular endothelial cells, morphological changes of the large arteries, abnormal renal function, insulin resistance, diabetes and pancreatic failure, and abnormal brain function also occur. There are few studies on the effects of prenatal and postnatal overnutrition on the metabolism of glycolipid in the female offspring, and few studies have been conducted to analyze the interaction between prenatal and postnatal overnutrition in the female offspring's adverse metabolic outcomes. Therefore, this topic is based on the construction of High-fat/fructose diet (HFFD) diet model in pre natal and postpartum. The effects of prenatal and postnatal overnutrition on the metabolism of glucose and lipid in female and female offspring mice were studied. The interaction between prenatal and postnatal overnutrition in the female offspring was discussed. In addition, this subject used bioinformatics to meta-analysis on the two groups of gene chip expression profiles. The differential expression genes of the liver of the female offspring of the prenatal high fat diet and the normal prenatal diet were screened. The gene chip expression data were analyzed in depth from two levels of gene and its encoding protein, which provided a theory for the further study of the molecular mechanism of the development of the fat generation Xie Qian in female offspring mice. Basic. Study methods: 1. unpregnant female parent mice were fed with Normal chow diet (NC) or HFFD feeding respectively. Two groups of female parent mice were given weight, fat content, blood glucose, insulin level, insulin tolerance test (Insulin tolerance test, ITT) and glucose tolerance test (Glucose tolerance test) and other tests. The female parent mice of group.2.HFFD and group NC were copulated with the male parent mice fed with NC; during pregnancy, group NC continued NC feeding and HFFD Group continued HFFD feeding; female offspring mice were randomly divided into NC or HFFD feeding mice randomly, which formed NC/NC, NC/HFFD, HFFD/NC, and groups. After diet / postpartum diet) these 4 different groups, after weaning, group NC/NC continued NC feeding, group NC/HFFD continued HFFD feeding, HFFD/NC Group continued NC feeding, and HFFD/HFFD Group continued HFFD feeding. The female offspring mice were fed on food intake, energy metabolism, body weight, fasting blood sugar, plasma insulin level, and plasma triglyceride (Triglyceride, TG) water. Level, plasma free fatty acid (Free fatty acid, FFA) level, plasma leptin level, ITT test, GTT experiment, fat content, liver weight, white adipose tissue (White adipose tissue, WAT) pathology, liver pathology, pancreatic beta cell morphology, glucose stimulated insulin release experiment (Glucose-stimulated insulin), and liver The protein and mRNA expression related to glycolipid metabolism in the tissue and pancreatic tissue were detected and analyzed by.3. in the Gene expression ominibus (GE0) database (Gene expression ominibus, GE0) to retrieve and download the data set of the liver gene expression profiles of the female offspring of the prenatal high fat diet and the normal prenatal diet; the data set was introduced into the data set. To BRB-Array Tools standardized treatment, sorting and analysis to obtain the differential expression genes of the liver of the female offspring of the prenatal high fat diet and the normal prenatal diet; the protein encoded by the differentially expressed genes were introduced into the STRING online analysis software, and the interwork network data of the protein encoded by the differentially expressed genes were obtained, and then introduced into the Cytoscape In software, the topology characteristics of the network and each node are calculated, and the key genes corresponding to the most Dalian pass protein are obtained; the The database for annotation visualization and integrated discovery, DAVID software is used to carry out the gene constitutive theory (Gene ontology, GO) function annotation and Kyoto gene and base. Kyoto Encyclopedia of Genes and Genomes, KEGG) pathway analysis. Results: 1. unpregnant female parent mice: the body shape of the group HFFD showed a more obvious obesity trend in group.HFFD, fat content, blood glucose and insulin levels were significantly higher than those of.HFFD group NC. The indexes of group C and group HFFD/NC were basically normal, and group HFFD/HFFD and NC/HFFD showed high weight, high fat content, high blood sugar and severe insulin resistance. Compared with the NC/HFFD group, the HFFD/HFFD group showed more obvious.HFFD/HFFD group and NC/HFFD plasma leptin level, and the plasma leptin level in HFFD/HFFD group was higher than that in NC/HFFD group. The mass, size and insulin release of beta cells in group HFFD/HFFD and group NC/HFFD were obviously abnormal. Compared with group NC/HFFD, the changes in HFFD/HFFD group were more obvious. When insulin was present, the phosphorylation of serine / threonine kinase (Serine/threoninekinase, Akt) in group NC/NC and HFFD/NC was significantly increased; and the NC/HFFD group Akt phosphorylation under insulin stimulation. Increase, but significantly lower than group NC/NC or group HFFD/NC; insulin stimulation did not induce Akt phosphorylation in group HFFD/HFFD to increase insulin in group.HFFD/HFFD and NC/HFFD, and the peroxisome proliferator activated receptor (Peroxidase body hyperplasia activated receptor gamma) and glucose phosphate phosphoric acid The expression of the enzyme (Glucose 6 phosphatase, G-6-Pase) protein and mRNA was significantly higher than that in the NC/NC group, and the expression of insulin, PGC-1 A and G-6-Pase protein and mRNA was higher in the HFFD/HFFD group than in the NC/HFFD group, and the glucose transporter and glucokinase protein and the table of the NC/HFFD group were higher than those in the NC/HFFD group. Compared with the NC/NC group, the HFFD/HFFD group was compared with the NC/HFFD group, and GLUT-2 and GCK protein and mRNA expressed 85 differentially expressed genes in the female offspring of the female offspring of the prenatal high fat diet and the normal prenatal diet, of which 38 were up and 47 were down regulated. The differential expression gene KEGG pathway analysis showed that there were 12 genes involved in Xie Tong. There were 4 genes involved in the Wnt signaling pathway and 4 genes involved in the myosinol metabolism pathway. This study found that 4 key genes were low density lipoprotein receptor related protein (Low-density lipoprotein receptor-related protein, LRP6), Prkd2, Kcnk13 and Notch1., respectively. 1. the excess nutrition before pregnancy could successfully induce the small parent The direct result of the fat accumulation in the female offspring, hyperglycemia, hyperinsulinemia, and beta cell dysfunction can further lead to the increase of leptin levels and leptin in the T2DM.3. cycle of the fat accumulation, hyperglycemia, hyperinsulinemia, insulin resistance and glucose tolerance.2.. Resistance is an important cause of fat accumulation in female offspring. The function of pancreatic beta cells is an important cause of insulin resistance and impaired glucose tolerance,.4.Akt phosphorylation and Akt function are inhibited; PGC-1 alpha, insulin and G-6-Pase expression are increased, glucose isogenesis and glycogen decomposition and activation; GLUT-2 and GCK expression decreased, glucose glycolysis decreased; The main reason for the abnormal metabolism of glycolipid in female offspring.5. female offspring is mainly caused by the overnutrition overnutrition in the female offspring. The simple prenatal nutrition can not directly cause the glucose and lipid metabolism disorder in the female offspring, but it has an important potential effect on the glycolipid metabolizing function of.6.LRP6, Prkd2, and Kcnk13. And Notch1 is the key gene.LRP6 in the differential expression gene of the liver of the female offspring of the prenatal high fat diet and the normal prenatal diet, which plays an important role in the effect of the high fat diet on the fat generation Xie Qian of the female offspring.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714
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本文编号：1901174