本文選題：營養(yǎng)過剩 + 高脂飲食��； 參考：《山東大學(xué)》2017年博士論文

【摘要】：背景:傳統(tǒng)觀念認(rèn)為,成人慢性疾病是在多基因遺傳易感性的基礎(chǔ)上,由包括不良生活方式在內(nèi)的環(huán)境因素所誘發(fā)的。1995年,Barker提出了胎兒編程(Fetal programming)假說,認(rèn)為生命早期的關(guān)鍵窗口對(duì)終身健康有"編程效應(yīng)",生命起始于胚胎和胎兒宮內(nèi)階段,宮內(nèi)環(huán)境是否正常關(guān)系到個(gè)體生命的成長、遠(yuǎn)期疾病的發(fā)生,甚至子孫后代的健康。2010年Motrenkoe又提出了胚胎源性疾病(Embryonic diseases)這個(gè)新概念,認(rèn)為配子和胚胎的發(fā)育異常有可能影響子代的健康狀態(tài),配子或胚胎對(duì)不利環(huán)境因素做出適應(yīng)性反應(yīng),最終可能引發(fā)子代成年后的糖尿病、肥胖、非酒精性脂肪肝(Non alcoholic fatty liver disease,NAFLD)和心腦血管疾病等疾病。大量研究證實(shí),產(chǎn)前(妊娠前和妊娠期)和產(chǎn)后(哺乳期和斷奶后早期)營養(yǎng)過剩都在子代糖脂代謝異常的發(fā)生和發(fā)展中起到了重要作用。圍妊娠期的飲食和生活習(xí)慣是親代和子代健康的重要決定因素。妊娠前超重和肥胖的婦女,流產(chǎn)、妊娠期糖尿病(Gestational diabetes mellitus,GDM)和子癇前期(Preeclampsia,PE)發(fā)病率明顯增加,高出生體重發(fā)生率明顯增加,子代成年后肥胖、Ⅱ型糖尿病(Type 2 diabetes mellitus,T2DM)和心血管疾病發(fā)病率也明顯增加。妊娠期過多的營養(yǎng)攝入是子代健康或疾病的獨(dú)立危險(xiǎn)因素,妊娠期過多的營養(yǎng)攝入與成年后高血壓、葡萄糖耐受性受損、非胰島素依賴型糖尿病(Non-insulin-dependent diabetes mellitus,NIDDM)或 T2DM、胰島素抵抗和肥胖的發(fā)病風(fēng)險(xiǎn)增加的有關(guān),除此之外,癌癥也與妊娠期營養(yǎng)過剩有關(guān),有研'究指出,高出生體重者更易罹患乳腺癌和白血病。有研究證實(shí),哺乳期高脂飲食的小鼠的子代成年后可患高血壓、血管內(nèi)皮細(xì)胞功能異常、大動(dòng)脈形態(tài)學(xué)改變、腎功異常、胰島素抵抗、糖尿病和胰腺衰竭等疾病,大腦功能異常也有發(fā)生。除此之外,子代斷奶后早期營養(yǎng)過剩也在將來代謝綜合征發(fā)病中起到重要作用。然而綜合考慮產(chǎn)前和產(chǎn)后營養(yǎng)過剩對(duì)雌性子代糖脂代謝影響的研究較少,也很少有研究綜合分析產(chǎn)前和產(chǎn)后營養(yǎng)過剩在雌性子代不良代謝結(jié)局中的相互作用關(guān)系。因此本課題通過構(gòu)建產(chǎn)前和產(chǎn)后高脂高果糖(High-fat/fructose diet,HFFD)飲食模型,研究產(chǎn)前和產(chǎn)后營養(yǎng)過剩對(duì)雌性親代小鼠和雌性子代小鼠糖脂代謝的影響及作用機(jī)制,探討產(chǎn)前和產(chǎn)后營養(yǎng)過剩在雌性子代不良結(jié)局中的相互作用關(guān)系。另外,本課題利用生物信息學(xué)方法對(duì)兩組基因芯片表達(dá)譜數(shù)據(jù)進(jìn)行薈萃分析,篩選產(chǎn)前高脂飲食和產(chǎn)前正常飲食的雌性子代小鼠肝臟的差異表達(dá)基因,從基因及其編碼蛋白質(zhì)兩個(gè)層面深入分析基因芯片表達(dá)譜數(shù)據(jù),為進(jìn)一步研究產(chǎn)前營養(yǎng)過剩對(duì)雌性子代小鼠脂代謝潛在影響的發(fā)生、發(fā)展的分子機(jī)制提供理論基礎(chǔ)。研究方法:1.未孕雌性親代小鼠分別常規(guī)飼料(Normal chow diet,NC)喂養(yǎng)或HFFD喂養(yǎng)。兩組雌性親代小鼠分別進(jìn)行體重、脂肪含量、血糖、胰島素水平、胰島素耐受實(shí)驗(yàn)(Insulin tolerance test,ITT)和葡萄糖耐受實(shí)驗(yàn)(Glucose tolerance test,GTT)等檢測(cè),并進(jìn)行統(tǒng)計(jì)學(xué)分析。2.HFFD組和NC組雌性親代小鼠與NC喂養(yǎng)的雄性親代小鼠交配;妊娠期,NC組繼續(xù)NC喂養(yǎng),HFFD組繼續(xù)HFFD喂養(yǎng);雌性子代小鼠隨機(jī)分組,分別由NC或HFFD喂養(yǎng)的親代小鼠隨機(jī)哺乳,這就形成了 NC/NC組,NC/HFFD組,HFFD/NC組和HFFD/HFFD組(產(chǎn)前飲食/產(chǎn)后飲食)這4個(gè)不同的組別;斷奶后,NC/NC組繼續(xù)NC喂養(yǎng),NC/HFFD組繼續(xù)HFFD喂養(yǎng),HFFD/NC組繼續(xù)NC喂養(yǎng),HFFD/HFFD組繼續(xù)HFFD喂養(yǎng)。四組雌性子代小鼠分別進(jìn)行攝食量、能量代謝、體重、空腹血糖、血漿胰島素水平、血漿甘油三酯(Triglyceride,TG)水平、血漿游離脂肪酸(Free fatty acid,FFA)水平、血漿瘦素水平、ITT實(shí)驗(yàn)、GTT實(shí)驗(yàn)、脂肪含量、肝臟重量、白色脂肪組織(White adipose tissue,WAT)病理、肝臟病理、胰腺β細(xì)胞形態(tài)、葡萄糖刺激的胰島素釋放實(shí)驗(yàn)(Glucose-stimulated insulin secretion,GSIS)以及肝臟組織和胰腺組織中與糖脂代謝相關(guān)的蛋白和mRNA表達(dá)等檢測(cè),并進(jìn)行統(tǒng)計(jì)學(xué)分析。3.在基因表達(dá)綜合數(shù)據(jù)庫(Gene expression ominibus,GE0)中檢索并下載兩組產(chǎn)前高脂飲食和產(chǎn)前正常飲食的雌性子代小鼠肝臟基因表達(dá)譜數(shù)據(jù)集;將數(shù)據(jù)集導(dǎo)入到BRB-Array Tools中標(biāo)準(zhǔn)化處理、整理、分析,獲得產(chǎn)前高脂飲食和產(chǎn)前正常飲食的雌性子代小鼠肝臟差異表達(dá)基因;向STRING在線分析軟件中導(dǎo)入差異表達(dá)基因所編碼蛋白,獲得差異表達(dá)基因所編碼蛋白的互作網(wǎng)絡(luò)數(shù)據(jù),再將其導(dǎo)入Cytoscape軟件中,計(jì)算網(wǎng)絡(luò)及各個(gè)節(jié)點(diǎn)的拓?fù)涮匦?得到最大連通度的蛋白所對(duì)應(yīng)的關(guān)鍵基因;利用基因注釋形象集成數(shù)據(jù)庫(The database for annotation visualization and integrated discovery,DAVID)軟件進(jìn)行基因本體論(Gene ontology,GO)功能注釋和京都基因與基因組百科全書(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析。結(jié)果:1.未孕雌性親代小鼠:HFFD組體形表現(xiàn)出更為明顯的肥胖趨勢(shì)。HFFD組體重、脂肪含量、血糖和胰島素水平均明顯高于NC組。HFFD組ITT和GTT也表現(xiàn)出明顯異常。2.雌性子代小鼠:NC/NC組和HFFD/NC組各項(xiàng)指標(biāo)基本正常;HFFD/HFFD組和NC/HFFD組表現(xiàn)出明顯高體重、高脂肪含量、高血糖和嚴(yán)重胰島素抵抗;與NC/HFFD組相比較,HFFD/HFFD組表現(xiàn)更為明顯。HFFD/HFFD組和NC/HFFD血漿瘦素水平明顯較高;HFFD/HFFD組血漿瘦素水平比NC/HFFD組更高。HFFD/HFFD組和NC/HFFD組β細(xì)胞質(zhì)量,大小和胰島素釋放情況明顯異常;與NC/HFFD組相比較,HFFD/HFFD組變化更加明顯。當(dāng)胰島素存在時(shí),NC/NC組和HFFD/NC組絲氨酸/蘇氨酸激酶(Serine/threoninekinase,Akt)磷酸化顯著增加;在胰島素刺激下NC/HFFD組Akt磷酸化增加,但顯著低于NC/NC組或HFFD/NC組;胰島素刺激不能誘導(dǎo)HFFD/HFFD組Akt磷酸化增加。HFFD/HFFD組和NC/HFFD組胰島素、過氧化物酶體增生激活受體丫協(xié)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 表達(dá)明顯高于 NC/NC 組;HFFD/HFFD組與NC/HFFD組相比,胰島素、PGC-1α和G-6-Pase蛋白和mRNA表達(dá)更高。HFFD/HFFD 組和 NC/HFFD 組葡萄糖轉(zhuǎn)運(yùn)蛋白(Glucose transport protein,GLUT)-2和葡萄糖激酶(Glucokinase,GCK)蛋白和mRNA表達(dá)明顯低于NC/NC組;HFFD/HFFD組與NC/HFFD組相比,GLUT-2和GCK蛋白和mRNA表達(dá)更低。3.產(chǎn)前高脂飲食和產(chǎn)前正常飲食的雌性子代小鼠肝臟差異表達(dá)基因85個(gè),其中上調(diào)基因38個(gè),下調(diào)基因47個(gè)。差異表達(dá)基因KEGG通路分析得出,有12個(gè)基因參與代謝通路,有4個(gè)基因參與Wnt信號(hào)通路,有4個(gè)基因參與磷酸肌醇代謝通路。本研究發(fā)現(xiàn)關(guān)鍵基因4個(gè),分別為低密度脂蛋白受體相關(guān)蛋白(Low-density lipoprotein receptor-related protein,LRP6)、Prkd2、Kcnk13和 Notch1。結(jié)論:1.妊娠前營養(yǎng)過剩可以成功誘導(dǎo)了親代小鼠脂肪蓄積、高血糖、高胰島素血癥、胰島素抵抗和葡萄糖耐量異常。2.產(chǎn)前和產(chǎn)后營養(yǎng)過剩的直接結(jié)果就是雌性子代小鼠脂肪蓄積,高血糖,高胰島素血癥,和β細(xì)胞功能障礙,可以進(jìn)一步導(dǎo)致其肥胖和T2DM。3.循環(huán)中瘦素水平上升和瘦素抵抗是雌性子代小鼠脂肪蓄積的重要原因。胰腺β細(xì)胞功能受累,是胰島素抵抗和糖耐量異常的重要原因。4.Akt磷酸化減少,Akt功能受到抑制;PGC-1 α、胰島素和G-6-Pase表達(dá)升高,糖異生和糖原分解活化;GLUT-2和GCK表達(dá)降低,葡萄糖酵解減少;這些都是雌性子代小鼠糖脂代謝異常的重要原因。5.雌性子代小鼠糖脂代謝障礙主要是主要是由產(chǎn)后營養(yǎng)過剩造成的。單純產(chǎn)前營養(yǎng)過剩不能直接造成雌性子代小鼠糖脂代謝障礙,但對(duì)其糖脂代謝功能有重要的潛在影響。6.LRP6,Prkd2,Kcnk13和Notch1是產(chǎn)前高脂飲食和產(chǎn)前正常飲食的雌性子代小鼠肝臟差異表達(dá)基因中的關(guān)鍵基因。LRP6在產(chǎn)前高脂飲食對(duì)雌性子代小鼠脂代謝潛在影響中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714
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本文編號(hào)：1901174