【摘要】：第一部分母親孕期高甘油三酯血癥通過(guò)重編程子代瘦素水平誘發(fā)成年期高血壓 目的：證實(shí)母親孕晚期高甘油三酯(TG)血癥可增加出生子代成年高血壓的發(fā)生風(fēng)險(xiǎn),探討瘦素(leptin)基因的甲基化改變?cè)谄渲邪l(fā)揮的作用。材料和方法：臨床隨訪不同出生體重的個(gè)體在學(xué)齡前期、學(xué)齡期和成年期的身高、體重和血壓的變化并留取部分人群血清檢測(cè)leptin水平。同時(shí)臨床調(diào)查2687例孕婦孕晚期的血脂數(shù)據(jù)和子代出生的基本信息,并留取母親血和臍帶血檢測(cè)血脂、脂肪酸以及l(fā)eptin等的水平；高脂喂養(yǎng)建立孕期高脂血癥大鼠模型,留取母親孕期血清檢測(cè)血脂水平,證實(shí)孕晚期存在高甘油三酯血癥。收集子代大鼠各個(gè)年齡段(胎鼠,3周,8周,6月,1年)的淋巴細(xì)胞和／或脂肪組織以及腎臟,分別檢測(cè)leptin基因的表達(dá)和腎小動(dòng)脈管壁厚度。用亞硫酸鹽測(cè)序方法檢測(cè)人臍血和外周血淋巴細(xì)胞leptin基因的表達(dá)和甲基化水平。同樣方法檢測(cè)胎鼠和3周大鼠的脂肪leptin基因的甲基化以及3周大鼠淋巴細(xì)胞的甲基化變化。利用胎鼠期間分離出來(lái)的骨髓來(lái)源的間充質(zhì)干細(xì)胞(MSCs)進(jìn)行深入的機(jī)制探討。MSc在成脂分化過(guò)程中加不同濃度的脂肪酸,檢測(cè)高濃度的脂肪酸對(duì)成脂分化過(guò)程中l(wèi)eptin基因表達(dá)和甲基化的作用。 結(jié)果：出生巨大兒的個(gè)體從兒童期一直到成年時(shí)期均表現(xiàn)出持續(xù)升高的血清leptin水平,且在成年期血壓明顯高于正常出生體重的成年個(gè)體,且30-50歲的高血壓發(fā)生率在巨大兒組明顯升高,盡管兩組之間的體重指數(shù)(BMI)無(wú)顯著差異。巨大兒和正常出生體重兩組的母親孕期血脂比較結(jié)果提示巨大兒組母血甘油三酯水平(mTG)顯著高于對(duì)照組。因此,我們?cè)俑鶕?jù)臨床調(diào)查的2687例孕晚期母親血TG值計(jì)算平均值為3.28mM,然后按照3.28mM把數(shù)據(jù)分為高母親血脂組(mTG3.28mM)和低母親血脂組(mTG3.28mM),結(jié)果發(fā)現(xiàn)高mTG組母親出生的子代臍帶血和學(xué)齡前期的外周血血清leptin水平均顯著升高,而且無(wú)論是出生時(shí)還是學(xué)齡前階段,血清leptin水平與母親血清TG水平呈顯著正相關(guān)。高脂喂養(yǎng)動(dòng)物出生子代自8周起體重已趨于同齡對(duì)照組大鼠,但是血壓顯著高于對(duì)照組,1年時(shí)出現(xiàn)明顯高血壓,伴有腎小動(dòng)脈管壁的明顯增厚。高脂母親出生的子代大鼠各個(gè)年齡段的血清leptin水平表現(xiàn)為持續(xù)升高,同時(shí)各個(gè)年齡段的脂肪組織和淋巴細(xì)胞leptin基因均持續(xù)顯著的高表達(dá)。高TG母親出生的新生兒和學(xué)齡前兒童的淋巴細(xì)胞leptin基因表達(dá)顯著高于對(duì)照組,同時(shí)伴隨著甲基化水平的顯著降低。與臨床結(jié)果一致,高脂喂養(yǎng)母親子代的胎鼠脂肪、3周齡的皮下脂肪和內(nèi)臟脂肪均出現(xiàn)leptin基因的高表達(dá)伴隨著低甲基化狀態(tài),同時(shí)3周齡子代大鼠的淋巴細(xì)胞leptin也呈現(xiàn)高表達(dá)和低甲基化。體外MSC誘導(dǎo)分化過(guò)程中加高濃度的脂肪酸可增加leptin基因的表達(dá)和促進(jìn)去甲基化酶TET1的高表達(dá),導(dǎo)致leptin基因的低甲基化。 結(jié)論：母親孕期高脂暴露可通過(guò)修飾leptin基因的甲基化影響其表達(dá)從而誘導(dǎo)子代成年高血壓的發(fā)生。 第二部分：母親孕期高甘油三酯血癥可通過(guò)調(diào)節(jié)leptin介導(dǎo)的膽固醇代謝通路增加子代高膽固醇血癥的風(fēng)險(xiǎn) 目的：探討母親孕期高甘油三酯血癥導(dǎo)致的持續(xù)升高的leptin水平是否通過(guò)調(diào)節(jié)膽固醇分解代謝的關(guān)鍵酶膽固醇7a-羥化酶(CYP7α1)的表達(dá),從而增加子代發(fā)生高膽固醇血癥的風(fēng)險(xiǎn)。 材料與方法：隨機(jī)電話隨訪2006-2007年在我院分娩出生的兒童250例,收集自愿回隨訪門(mén)診體檢的49例學(xué)齡前期兒童(3-6歲)的血清檢測(cè)血清leptin、血脂四項(xiàng)等水平,同時(shí)回顧性調(diào)查其母親孕期的血脂水平和出生相關(guān)資料。利用孕期高脂喂養(yǎng)大鼠模型進(jìn)一步驗(yàn)證高脂子代3周齡大小時(shí)血清leptin以及血脂水平,同時(shí)用化學(xué)發(fā)光法檢測(cè)3周齡子代大鼠肝臟組織勻漿上清血脂水平。Real-time PCR和Western分別用于檢測(cè)3周子代大鼠肝臟組織膽固醇代謝關(guān)鍵酶的表達(dá)。利用體外培養(yǎng)肝細(xì)胞系HepG2進(jìn)行機(jī)制的研究,觀察leptin是否可濃度依賴性的調(diào)節(jié)通路因子LEPR-JAK2-STAT3的表達(dá),同時(shí)調(diào)控CYP7α1的表達(dá)；siRNA靶向干擾轉(zhuǎn)錄因子后進(jìn)一步證實(shí)leptin刺激后通過(guò)STAT3發(fā)揮調(diào)節(jié)作用；免疫共沉淀(CHIP)試驗(yàn)證實(shí)轉(zhuǎn)錄因子STAT3可結(jié)合到CYP7α1基因啟動(dòng)子區(qū)域發(fā)揮調(diào)控作用。 結(jié)果：高mTG組出生子代兒童血清leptin、膽固醇水平顯著高于mTG組,兒童血清leptin水平與膽固醇水平呈顯著正相關(guān)。高脂喂養(yǎng)大鼠出生的子代3周齡時(shí)血清leptin和膽固醇水平也呈現(xiàn)顯著升高,肝臟組織勻漿上清總膽固醇水平在高脂子代組顯著高于正常喂養(yǎng)子代組,同時(shí)伴有膽固醇降解關(guān)鍵酶CYP7α1的顯著下調(diào)。體外細(xì)胞實(shí)驗(yàn)結(jié)果顯示：隨著leptin濃度的不斷增加,通路因子LEPR、 JAK2、STAT3表達(dá)濃度依賴性上調(diào),而高濃度的leptin可下調(diào)CYP7α1的表達(dá)。靶向干擾STAT3后,leptin刺激下CYP7α1表達(dá)不再濃度下調(diào),CHIP試驗(yàn)結(jié)果進(jìn)一步提示轉(zhuǎn)錄因子STAT3可結(jié)合CYP7α1基因啟動(dòng)子區(qū)域發(fā)揮調(diào)控作用。 結(jié)論：母親孕期高甘油三酯血癥出生子代兒童期高膽固醇血癥風(fēng)險(xiǎn)增加,孕期高脂誘導(dǎo)的子代持續(xù)高leptin水平可通過(guò)LEPR-JAK2-STAT3通路調(diào)控膽固醇降解的關(guān)鍵酶CYP7α1基因的表達(dá)。 第三部分：孕期高脂暴露誘發(fā)的子代脂質(zhì)代謝異常的傳代機(jī)制研究 目的：探討親代母親孕期高脂飲食是否對(duì)子一代(F1)脂質(zhì)代謝產(chǎn)生影響以及這種不良影響是否能通過(guò)配子傳至子二代(F2) 材料與方法：建立孕期高脂喂養(yǎng)(HFD)的大鼠模型,觀察出生的F1代在恢復(fù)正常喂養(yǎng)后各年齡段的體重和脂質(zhì)代謝,再用F1代分別與正常異性大鼠交配以及高脂子代組內(nèi)交配,觀察HFD-F1♂以及HFD-F1♀出生的F2代的各年齡段體重以及脂質(zhì)代謝變化。Real-time PCR方法篩查F1代、F2代肝臟和F1精子中脂代謝相關(guān)的印記基因,篩選出母源印記基因Igf2和父源印記基因Igf2r做進(jìn)一步的機(jī)制研究。再用亞硫酸鹽修飾測(cè)序方法分別檢測(cè)F2成年肝臟中這兩個(gè)印記基因的甲基化改變,來(lái)探討親代異常宮內(nèi)環(huán)境暴露影響子代的脂質(zhì)代謝的傳代機(jī)制。結(jié)果：高脂喂養(yǎng)出生的F1代3周內(nèi)體重顯著高于正常對(duì)照組,但是8周起兩組之間無(wú)顯著差異。HFD-F1在3周時(shí)出現(xiàn)明顯的血脂異常,表現(xiàn)為T(mén)G, TC, HDL和LDL水平顯著高于對(duì)照F1,8周和12周兩組間無(wú)明顯差異,但是HFD-F1在6月和1年時(shí)出現(xiàn)明顯的TG的升高,1年的時(shí)候同時(shí)出現(xiàn)LDL的升高和HDL的下降。盡管F1代恢復(fù)正常飲食,且受孕前和孕期均無(wú)高脂血癥,但是HFD-F1(?)出生的F2代在8周、12周及6月分別表現(xiàn)為血TG水平的顯著升高,且在12周和6月還伴隨著LDL的升高；與此不同的是,HFD-F1(?)出生的F2與對(duì)照組間差異不明顯。我們分別篩查了F1和F2肝臟中脂代謝相關(guān)的基因基因,發(fā)現(xiàn)母源印記基因Igf2和父源印記基因Igf2r的甲基化持續(xù)的高表達(dá),同時(shí)在F1精子和F2胎肝上驗(yàn)證這兩個(gè)基因均呈現(xiàn)高表達(dá)。因此我們檢測(cè)了F2肝臟中的印記基因Igf2和Igf2r的甲基化改變,結(jié)果提示HFD-F1(?)出生的F2成年肝臟的Igf2出現(xiàn)2個(gè)位點(diǎn)的低甲基化,Igf2r基因的總甲基化顯著下降,伴有5個(gè)位點(diǎn)的低甲基化。 結(jié)論：親代孕期高脂飲食可致子一代脂質(zhì)代謝異常,異常的脂代謝變化可通過(guò)子一代的精子傳至子二代,導(dǎo)致子二代成年期血脂代謝異常。
[Abstract]:The first part of mother's hypertriglyceridemia induced a one-year high blood pressure by reprogramming the level of leptin in the offspring Objective: To confirm that the high triglyceride (TG) in the mother trimester of pregnancy can increase the incidence of adult high blood pressure in the offspring. To explore the role of leptin in the methylation of leptin gene Materials and Methods: The change of height, body weight and blood pressure of the individual with different birth weight in the pre-school period, the normal period and the adulthood, and the serum detection of leptin in some of the population Ping. At the same time,2687 pregnant women were surveyed with the basic information of blood lipid data and the birth of the offspring, and the levels of blood fat, fatty acid and leptin were detected by mother blood and umbilical cord blood. The model of hyperlipidemic rats during pregnancy was established by high-fat feeding. It is confirmed that high triglyceride blood is present in the late stage of the pregnancy The expression of the leptin gene and the wall thickness of the renal arterioles were detected by collecting the lymphocytes and/ or the adipose tissue and the kidney of each age group (fetal rat,3-week,8-week,6-month,1 year) of the offspring rats. degree. Detection of the expression and methylation of leptin in human umbilical cord blood and peripheral blood lymphocytes by a sulfite sequencing method Ping. The methylation of the leptin gene in the fetal and 3-week rats and the methylation of the 3-week rat lymphocytes were also detected by the same method. In-depth mechanism of bone marrow-derived mesenchymal stem cells (MSCs) isolated from the fetal rat The effect of high concentration of fatty acid on the expression and methylation of leptin in the process of adipocyte differentiation was studied by adding different concentrations of fatty acid to the process of adipocyte differentiation. Results: The level of serum leptin, which was continuously elevated from childhood to adulthood, was observed in the individual from childhood to adulthood, and the blood pressure in adulthood was significantly higher than that of the normal birth weight, and the incidence of hypertension in the 30-50 year of age was in the large group. The significant increase, although the body mass index (BMI) between the two groups was not significant The results showed that the serum triglyceride level (mTG) was significantly higher in the mothers with large and normal birth weight. In the control group, therefore, we calculated the mean value of the maternal blood TG value of 3.28 mM and then divided the data into the high-mother blood-lipid group (mTG3.28 mM) and the low-mother blood-lipid group (mTG3.28) according to the 2687 cases of the clinical investigation. The results showed that the level of serum leptin in the peripheral blood of the mothers of the high mTG group was increased significantly, and the level of leptin and the level of TG in the mother serum were significantly higher either at the time of birth or in the pre-school stage. The weight of the birth of the high-fat-fed animal was increased from 8 weeks to the control group in the same age, but the blood pressure was significantly higher than that in the control group. The level of leptin in each age group of the offspring of the high-fat mother was increased continuously, while the leptin gene of the adipose tissue and the lymphocytes of all age groups continued to be significant. High expression. The expression of lepitin gene in the neonatal and pre-school children born by the high TG mother was significantly higher than that of the control group, accompanied by a significant increase in the level of methylation. In the same time, the high expression of leptin was associated with the low methylation status of the fetal rat fat,3-week-old subcutaneous fat and visceral fat of the high-fat-fed mother, and the high expression and the low level of the lymphocyte letin in the 3-week-old offspring were also presented. The high concentration of fatty acid in the induction and differentiation of MSC in vitro can increase the expression of leptin gene and promote the high expression of the demethylase TET1, leading to the low leptin gene. Conclusion: The high-fat exposure of mother can influence its expression by modifying the methylation of leptin gene, so as to induce the adult height of the offspring. The second part: hypertriglyceridemia in the mother's pregnancy can increase the offspring's height by adjusting the leptin-mediated cholesterol metabolism pathway cholesterol Objective of the study of the risk of hypertriglyceridemia: to explore whether the level of leptin, which is caused by hypertriglyceridemia during pregnancy, is increased by regulating the expression of the key enzyme-cholesterol 7a-hydroxylase (CYP7-1) in cholesterol catabolism, thus increasing the generation of offspring. The risk of hypercholesteremia. Materials and Methods:250 cases of children born in our hospital from 2006 to 2007 were followed up by random telephone, and the serum of 49 pre-school children (3-6 years) who received a voluntary follow-up visit were collected. The levels of tin, blood lipid, and the like, and the mother's pregnancy were retrospectively investigated. The serum leptin and blood lipid level of 3-week-old offspring of high-fat children were further verified by high-fat-fed rat model during pregnancy, and the liver of 3-week-old offspring was detected by the chemiluminescence method. The blood lipid level of the homogenate of the dirty tissue was detected by Real-time PCR and Western blot. The expression of the key enzyme of cholesterol metabolism was studied. The expression of LEPR-JAK2-STAT3 was regulated by the mechanism of the in vitro culture of the liver cell line HepG2, and the expression of CYP7-1 was also regulated. STAT3 plays a regulating role; the immune co-precipitation (CHIP) test confirms that the transcription factor STAT3 can bind to the CYP7-1 gene The results showed that the serum leptin and cholesterol in the children with high mTG were significantly higher than those in the mTG group, and the serum leptin in the children was higher than that of the mTG group. There was a significant positive correlation between the level of cholesterol and the level of cholesterol. The level of serum leptin and cholesterol in high-fat-fed rats increased significantly, and the total cholesterol level in the liver tissue homogenate was significantly higher in the high-fat offspring than in the normal-feeding progeny group, accompanied by a cholesterol-degrading effect. The results of in vitro cell experiment show that the expression of LEPR, JAK2 and STAT3 in the pathway is up-regulated with the increase of leptin concentration, while the high concentration of lepti N The expression of CYP7-1 was downregulated. After the target of STAT3, the expression of CYP7-1 under the stimulation of leptin was no longer reduced, and the results of CHIP test further suggested that transcription factor STAT3 could bind to CYP7-1. Conclusion: The risk of high-cholesteremia in the children with hypertriglyceridemia during pregnancy is increased, and the high-fat-induced high-fat content of the offspring during pregnancy can be regulated by the LEPR-JAK2-STAT3 pathway. Expression of the key enzyme CYP7-1 gene in the third part: high-fat exposure during pregnancy induced Objective: To investigate the effect of high-fat diet on the lipid metabolism of the subgeneration (F1) in the parent mother during pregnancy, and to study the effect of the high-fat diet on the lipid metabolism of the offspring (F1). whether the adverse effect can be passed through the gamete to the sub-second generation (F2) material and Methods: The rat model of high-fat feeding (HFD) during pregnancy was established. The body weight and lipid metabolism of the F1 generation after normal feeding were observed, and then the F1 generation was used to mate with the normal sex rats and the high-fat children, and the HFD-F1 mice and the HFD-F1 mice were observed. The weight of each age group and the change of lipid metabolism in the F2 generation of the birth. The real-time PCR method is used to screen the imprinting gene related to the lipid metabolism in the F1 generation, the F2 generation liver and the F1 sperm to screen the maternal imprinting gene Igf2. And the methylation changes of the two imprinted genes in the F2 adult liver are respectively detected by the sulfite-modified sequencing method to explore the parent. The results showed that the body weight was significantly higher in the first 3 weeks of high-fat feeding. There was no significant difference between the two groups in the normal control group, but there was no significant difference between the two groups at 8 weeks. The results showed that the levels of TG, TC, HDL and LDL were significantly higher than that of control F1,8 and 12 weeks, but there was no significant difference between the two groups at 6 and 1. At the same time, there was a rise in LDL and a decrease in HDL. Although the F1 generation returned to normal diet and was subject to pregnancy There was no hyperlipoidemia before and during pregnancy, but the F2 generation of HFD-F1 (?) was shown to be a significant increase in the level of blood TG at 8 weeks,12 weeks and 6 months, and was accompanied by 12 and 6 months L the elevation of the dl; unlike this, the hfd -F1 (?) was not significantly different from the control group. We screened the gene gene related to lipid metabolism in the liver of F1 and F2, respectively, and found that the methylation of the maternal imprinting gene Igor and the parent-source imprinting gene Igf2r was continued high, while at F1 The two genes were demonstrated to be highly expressed on both the sperm and the F2 fetal liver, so we detected the imprinting gene Igor and Ig in the F2 liver f The results suggested that the HFD-F1 (?) was a low-methylation, Igf2r gene in 2 sites in the F2 adult liver. Conclusion: The high-fat diet in the parent pregnancy can cause the abnormal lipid metabolism of the first generation, and the abnormal lipid metabolism can be changed through the sub-generation.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R714.256

【共引文獻(xiàn)】

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