【作者】 吕娟秀；

【导师】 徐智策； 茅彩萍；

【作者基本信息】 苏州大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 背景与目的高血压病为临床常见病与高发病,其发病机理仍未完全阐明。肾素血管紧张素系统参与了机体水钠代谢。心脏、肾脏及大脑是高血压危害的重要靶器官。目前认为母体子宫内的环境因素与其成年子代的高血压发生有重要关系。妊娠期高盐因素可能影响胚胎肾素血管紧张素系统的发育进而造成高血压相关的靶器官的发育问题。鉴于上述问题,本课题研究母体高盐饮食对成年子代高血压的影响。主要研究:1、妊娠期母鼠给于8%氯化钠饮食,观察子宫内胎鼠的血管紧张素系统发育。2、血管紧张素系统对水钠代谢的调节。3、母体高盐饮食对子宫内胎鼠的高血压相关的靶器官（心,脑,肾）的发育的影响。4、ATtb受体基因的表观遗传学机制参与的母体高盐饮食对胎鼠心脏的作用。方法1.检测:每天称量母鼠体重,摄食量,饮水量,排尿量;检测血压,尿钠离子浓度,检测母鼠和胎鼠的血浆渗透压和血液钠离子的浓度。2.用放射免疫法检测母鼠和胎鼠血液中血管紧张素Ⅱ和醛固酮的浓度以及胎鼠心脏和肾脏组织中血管紧张素Ⅱ浓度。3.用透射电镜方法观察胎鼠的心脏及肾脏组织在母体高盐因素的影响下超微结构的变化。4.用western blot和荧光定量PCR的方法检测胎鼠肾脏、心脏及脑内AT₁和AT₂受体的蛋白和mRNA的表达,检测胎鼠肝脏及脑内血管紧张素原mRNA的表达。5.体外原代细胞培养胎鼠心肌细胞及肾脏系膜细胞,流式细胞仪分析心肌细胞及肾脏系膜细胞的细胞周期,并研究AngⅡ对培养细胞的影响。6.用焦磷酸测序法分析胎鼠心脏AT_1b受体基因的甲基化模式。结果1.高盐饮食组的母鼠的饮水量、24小时尿量和尿中钠离子浓度增多,但摄食量及体重没有改变;母鼠在高盐饮食的第二周开始有血压增高的趋势,但没有显著的统计学差异。GD21高盐饮食组母鼠和胎鼠血浆渗透压和血液钠离子浓度没有变化。2.高盐饮食组母鼠和胎鼠血浆AngⅡ和醛固酮浓度降低,胎鼠心脏和肾脏组织AngⅡ浓度升高。3.高盐饮食组胎鼠心脏超微结构的变化:心肌细胞线粒体嵴减少、空泡化,心肌纤维排列紊乱,部分心肌纤维原消失;肾脏超微结构的变化:肾脏足突减少,足突融合,基底膜裸露,部分基底膜消失。4.胎鼠心脏组织AT_1a,AT_1b mRNA表达增多,AT₁受体蛋白表达增多,AT₂受体mRNA和蛋白表达没有改变;胎鼠肾脏组织AT_1b mRNA表达增多,AT₁受体蛋白表达增多,AT_1a mRNA表达无变化,AT₂受体的mRNN和蛋白的表达没有改变;胎鼠脑组织AT_1a,AT_1b,AT₂受体mRNA表达增多,血管紧张素原mRNA表达降低,AT₁,AT₂受体蛋白表达增多;胎鼠肝脏血管紧张素原mRNA表达增高。5.体外原代细胞培养发现:高盐饮食组胎鼠心肌细胞和肾脏系膜细胞增殖能力增强。AngⅡ刺激心肌细胞和肾脏系膜细胞增殖,AT₁受体阻断剂（losartan）能部分翻转AngⅡ对细胞生长的刺激作用。AT₂受体阻断剂（PD123319）不能抑制AngⅡ对细胞生长的刺激作用。6.高盐饮食组胎鼠心脏AT_1b受体启动子区域-1499,-1511,-1517,-1522,-1566序列甲基化程度下调。结论:1.妊娠期间母体高盐饮食使母鼠的饮水及肾脏排水排钠增多以保证母鼠和胎鼠的水钠代谢平衡。在摄入高盐的条件下,母鼠的血压有逐渐升高趋势。2.妊娠期间母体高盐饮食抑制了母鼠和胎鼠外周血管紧张素系统。3.妊娠期间母体高盐饮食影响了胎鼠心脏和肾脏的发育。4.妊娠期间母体高盐饮食可影响胎鼠心脏、肾脏、脑和肝脏血管紧张素系统发育。5.胎鼠心脏和肾脏局部高浓度AngⅡ可刺激心肌细胞和肾脏系膜细胞增殖。AT₁受体介导了AngⅡ对心肌细胞和肾脏系膜细胞生长的刺激作用。6.AT_1b受体DNA甲基化的改变可部分解释母体高盐饮食对子宫内胎鼠的心脏作用.更多还原

【Abstract】 Background:Hypertension is a common cardiovascular disease.The origin and biological mechanisms of fetal programming has attracted attention.The ’foetal origins of adult disease’ hypothesis is important to preventative medicine.Epidemiological studies have indicated that susceptibility of hypertension and cardiovascular diseases may result from high salt diet.The mechanisms underlying programming are likely to be multifactorial.However,further studies have shown that programming of hypertension may occur in fetal origins and related to changes of the renin angiotensin systemPurpose:The present study was proposed:1.To explore the development of the fetal RAS following metemal high salt diet.2.To determine the relationship between the fetal RAS and body fluid balance.3.To identify changes in the fetal heart,kidney, and brain induced by meternal high salt diet.4.To determine the effect of intrauterine high salt on the fetal heart and DNA methylation.Method:1.Pregnant rats were randomized to a normal-salt（1%） or high-salt（8%） diet from gestational day（GD） 3 to GD21,there were 8 rats in each group.Rats were placed in metabolic cages,food and water intake,urine volume,and urinary sodium, and maternal and fetal plasma osmolarity,and sodium concentration were measured.2. Transmission electron microscope was used for analysis of the fetal heart and kidney.3. Plasma and tissue AngⅡ,and aldosterone were measured by radioimmunoassay.4.The component of RAS in the fetal heart,kidney,brain and liver were measured by real time PCR and western blot.5.Cell cycle was estimated for the proliferation of the fetal heart and mesangial cells.6.Pyrosequencing analysis for the DNA methylation of the AT_1b promoter in the fetal heart.Results:1.Water intake,urine volume and urine sodium excretion were increased by high salt diet in pregnant rats.There was no significantly difference in plasma Na⁺ and osmolarity in maternal and fetal rats between normal diet and high salt diet.2. Maternal salt loading elicited ultrastructural changes in the fetal heart and kidney.3. High salt diet caused plasma AngⅡand aldosterone concentrations decreased in both mothers and fetuses.AngⅡconcentration was increased in the fetal myocardium and kidney in high salt diet.4.AT₁ receptor protein was increased in the fetal heat and kidney induced by high salt diet,AT₁,and AT₂ receptor protein were increased in the fetal brain by high salt diet;high salt diet caused the changes in the fetal RAS as following:AT_1a and AT_1b mRNA were increased in the heart,AT_1b mRNA was increased in the kidney,AT_1a,AT_1b,and AT₂ mRNA were increased the brain,angiotensinogen mRNA was increased in the liver and dereased in the brain.5.AngⅡstimulated the fetal heart and mesangial cell proliferation;the effect of AngⅡon cellular growth is primarily mediated by the AT₁ receptor.6.DNA hypomethylation of AT_1b promoter resultd from maternal high salt diet.Conclusion 1.Maternal high salt diet had adverse effects on the development of the fetal heart and kidney.2.The local RAS activation induced by maternal high salt diet contributed to the change of the heart and kidney.3.Epigenetic mechanisms contributed to the prenatal programming of hypertension by maternal high salt diet.更多还原