【作者】 管俊昌；

【导师】 徐智策； 茅彩萍；

【作者基本信息】 苏州大学 ， 病原生物学， 2009， 博士

【摘要】 研究背景:妊娠期多种原因如过度活动、发热、出血、呕吐及腹泻等均可造成孕妇机体脱水。脱水影响机体体液中水和电解质平衡,导致细胞内外失水。肾素血管紧张素系统(renin-angiotensin system, RAS)是调控体液平衡的重要内分泌系统,在调节机体心血管活动、神经内分泌、饮水饮盐行为等方面均起着重要作用。大量研究表明机体脱水可导致体液失衡及影响RAS,但有关妊娠期脱水对胎儿RAS及心脏、肾脏发育的影响尚不清楚。Barker理论是目前国际上有关胎源性疾病的研究热点,大量流行病学及实验证据表明:宫内应激所导致“印迹”效应可“程序化”影响成年后心血管疾病(如高血压等)的发生,并发现RAS在高血压发病过程中起重要作用。然而,妊娠期母鼠脱水是否对成年子代的RAS产生印迹效应,是否影响子代RAS对体液平衡及心血管功能的调控,目前国内外尚未见报道。因此,本实验研究妊娠晚期母鼠脱水对胎鼠RAS和心脏、肾脏发育的影响,并研究脱水对成年子代大鼠RAS的印迹效应及其功能的影响。第一部分妊娠晚期母鼠脱水对胎儿及其RAS发育的影响目的:妊娠晚期母鼠脱水对胎儿的调控因素RAS及心脏、肾脏发育的影响。方法:妊娠晚期母鼠脱水三天后,称量胎鼠体重、心重、肾重及其干重;血气分析仪检测母、胎鼠的血气指标和电解质,用渗透压仪检测血浆渗透压;用放射免疫法检测母、胎鼠血液中血管紧张素(angiotension, Ang)Ⅰ、AngⅡ、血管加压素(vasopressin,VP)和醛固酮(aldosterone,ALD)的浓度;用透射电镜方法检测胎鼠心、肾脏组织超微结构,并用激光多谱勒检测肾脏表面血流;用western blot和荧光定量PCR的方法检测胎鼠心脏、肾脏中血管紧张素受体(ATR)的蛋白和mRNA水平,并检测胎鼠肝脏中血管紧张素原(angiotensinogen, ATG)的mRNA水平。结果:妊娠晚期母鼠脱水可使胎鼠体重、心脏和肾脏的重量明显降低,胎鼠血钠、红细胞压积及血浆渗透压增加,胎鼠血液中AngI、AngⅡ、VP和ALD的浓度明显增加,及肝脏中ATG的mRNA水平显著增加。妊娠晚期脱水可导致胎鼠心脏及肾脏超微结构的改变,明显增加胎鼠心脏中AT2R的mRNA和蛋白水平,但不影响AT1aR及AT1bR的mRNA和AT1R的蛋白水平;可明显增加胎鼠肾脏中AT1R与AT2R的mRNA和蛋白水平,并减少肾血流量。结论:妊娠晚期脱水可明显改变胎鼠体内的RAS水平,并影响胎鼠心脏和肾脏的发育。第二部分妊娠晚期母鼠脱水对成年子代RAS的“印迹”效应及其功能的影响目的:研究妊娠晚期母鼠脱水对成年子代大鼠体内RAS水平的影响及其“印迹”效应,并研究妊娠晚期母鼠脱水对成年子代大鼠RAS调控体液平衡及心血管系统功能的影响。方法:用妊娠晚期母鼠脱水组及对照组成年子代,检测子代大鼠体重、心脏和肾脏重量。在基础状态下,用血气分析仪检测成年子代大鼠血气指标和电解质,用渗透压仪检测血浆渗透压;用放射免疫法检测血液中AngI、AngⅡ、VP和ALD的浓度;检测成年子代大鼠心血管系统对PD123319、Losartan及AngII的应答;检测离体胸主动脉及肠系膜动脉对血管舒缩物质的反应;用western blot和荧光定量PCR的方法检测心脏及肾脏中AT1R和AT2R的蛋白和mRNA水平,并检测肝脏中ATG的mRNA水平。在脱水应激下,检测成年子代大鼠体内RAS组分的浓度,并检测脱水对成年子代大鼠体液平衡、心血管系统及饮水饮盐行为的影响。结果:妊娠晚期母鼠脱水组雌、雄性成年子代大鼠的体重、心脏及肾脏的重量、血气指标及电解质、血液中AngI、AngⅡ、VP和ALD的浓度与对照组比较无差异,但脱水组成年子代大鼠肝脏中ATG mRNA水平及心脏、肾脏中AT1R与AT2R的mRNA和蛋白水平均明显不同于对照组。与对照组比较,脱水组雄性成年子代大鼠对AngⅡ、Losartan及PD123319的反应均明显增强,而雌性子代无差异性,但雌、雄性成年子代大鼠的压力反射敏感性均明显降低。脱水组成年子代大鼠的胸主动脉及肠系膜动脉对AngⅡ的收缩血管反应均明显增强。成年期脱水48h后,脱水组的雌、雄性成年子代鼠血气指标及电解质与对照组比较无差异性,但血浆中AngⅡ、VP和ALD的浓度明显高于对照组,饮盐量和血压也明显升高。结论:妊娠晚期母鼠脱水对成年子代大鼠的RAS有“印迹”效应。脱水组雌、雄性成年子代大鼠对心血管系统的调控存在性别差异,压力反射敏感性降低可能是子代心血管系统应答“程序化”效应的共同机制。脱水组成年子代大鼠的血管对AngⅡ升高血压的敏感性增强,提示其发生高血压等心血管疾病的危险性明显升高。脱水组成年子代大鼠在脱水刺激时,AngⅡ、ALD及VP浓度升高更明显,这与饮盐量及血压的显著增加可能相关,表现为患高血压等心血管疾病的“易感性”提高。第三部分妊娠晚期母鼠脱水对雄性成年子代大鼠心脏和肾脏蛋白质组影响的初探目的:妊娠晚期母鼠脱水对雄性成年子代大鼠心脏和肾脏蛋白质组表达影响。方法:采用双向电泳技术检测对照组及脱水组雄性成年子代大鼠心脏和肾脏中蛋白质组表达的差异。结果:雄性成年子代大鼠心脏和肾脏组织中蛋白质分布模式在对照组和实验组之间基本一致。对照组和实验组心脏中获得的平均蛋白质斑点数分别为603±25及577±32个,差异点数为33个;而肾脏中平均蛋白质斑点数分别为975±30及1107±42个,差异点数为38个。结论:初步建立了分辨率较高、重复性较好的正常和脱水组成年子代大鼠心脏与肾脏中总蛋白质双向电泳图谱。妊娠晚期母鼠脱水影响雄性成年子代大鼠心脏和肾脏中蛋白质组的表达。更多还原

【Abstract】 Background: Women may face mild to severe dehydration due to exercise, fever, hemorrhage, vomiting, and diarrhea during pregnancy. Water deprivation (WD) is a potent stimulation to affect body fluid balance, and can lead to both extracellular and intracellular dehydration. Renin-angiotensin system (RAS), as an endocrine system, plays a role in regulating body fluid balance, cardiovascular system, neuroendocrine activity, and water and salt intake. A lot of evidence suggests that WD could induce changes in body fluid balance and RAS components in adults. However, it is largely unknown of effects of WD during pregnancy on the development of fetal RAS, and of the heart and kidney.Barker theory hypothesizes diseases in fetal origins. There is now a body of substantial epidemiological and experimental evidence showing that intrauterine stress may program cardiovascular diseases such as hypertension in late life. Previous studies suggested that the RAS may play a critical role in pathophysiological progress of hypertension induced by adverse environment during fetal development. It is unknown whether exposure to prenatal WD can affect the RAS in its regulation of body fluid balance and cardiovascular system in the offspring. Hence, this project studies the effect of maternal WD during late term on the development of RAS, heart, kidney in both fetuses and offspring.PART 1 Effects of maternal WD during late gestation on development of fetus and fetal RASObjective: To determine the development of the RAS, heart and kidney of the fetus exposure to WD at late term.Methods: After three days of maternal WD, fetal body, heart, and kidney were weighted, and fetal blood gases and electrolytes were determined with a Nova analyzer, plasma osmolality was determined with an advanced digmatic osmometer. Both fetal and maternal plasma angiotensin (Ang)Ⅰ, AngⅡ, vasopressin (VP) and aldosterone (ALD) concentrations were measured by radioimmunoassay. The ultrastructures of fetal heart and kidney were detected by a transmission electron microscope and the renal blood flow was detected by a Laser Doppler. Protein and mRNA of AngⅡreceptors (ATR) in the fetal heart and kidney were determined with western-blot or real-time PCR, and ATG mRNA in the fetal liver was also determined.Results: Prenatal exposure to WD at late term significantly decreased weight of the fetal body, heart and kidney, and increased fetal plasma Na+, hematocrit and osmolality. Fetal liver ATG mRNA, plasma AngⅠ, AngⅡ, VP, and ALD concentrations were also increased following WD. Maternal WD during late gestation changed the ultrastructures in the fetal heart and kidney, and altered the expression of both protein and mRNA of ATR in the fetal kidney and heart. Fetal renal blood flow was reduced.Conclusion: Three days of maternal WD during late gestation significantly changed the concentrations of fetal RAS components, and affected the development of fetal heart and kidney.PART 2 Imprinting effect and function of RAS of adult offspring exposure to maternal WD during late gestationObjective: To determine the effect of maternal WD during late gestation on the RAS and its roles in regulation of body fluids and cardiovascular system in offspring. Methods: The offspring in both the control and WD groups were used. The adult offspring body, heart, and kidney were weighted. Blood gases and electrolytes were determined with a Nova analyzer, plasma osmolality was determined with an advanced digmatic osmometer, plasma AngⅠ, AngⅡ, VP, and ALD concentrations were measured by radioimmunoassay. Cardiovascular responses to intravenous injection of PD, losartan, and AngⅡ, and contractility of the thoracic aorta and mesenteric artery were determined. Protein and mRNA of ATR were measured with western blot and real-time PCR in both the heart and kidney of offspring, and ATG mRNA in fetal liver was also determined. The effect of WD on the RAS, body fluid balance, cardiovascular system, and both salt and water intake, offspring was determined.Results: Maternal WD during late gestation did not change the weights of body, heart and kidney, the blood gases and electrolytes, the concentrations of plasma AngⅠ, AngⅡ, VP and ALD in adult offspring, but maternal WD during late gestation significantly changed the ATG mRNA levels in liver, and both ATR mRNA and proteins in both heart and kidney in adult offspring. Cardiovascular responses to injection of PD, losartan and AngⅡwere notably enhanced in male not female adult offspring in WD group compared to control, but baroreflex sensitivity was notably attenuated in both male and female offspring. Compared to control, the ability of both thoracic aorta and mesenteric artery to vasoconstriction to AngⅡwas enhanced in adult offspring of WD group. After 48h of WD again, blood gases and electrolytes had no difference in adult offsprings between control and WD group, but the concentrations of plasma AngⅡ, VP and ALD, salt intake and blood pressure were significantly elevated in adult offsprings in WD group than those of control.Conclusion: Maternal WD during late gestation showed chronic influence on the RAS in the offspring. Cardiovascular responses to the stimulation following exposure to prenatal WD showed difference that was gender-dependent. The decrease of baroreflex sensitivity may be the mechanism for changes in the cardiovascular responses. Blood pressure and vasoconstriction to AngⅡwere significantly increased in the adult offspring exposure to prenatal WD. Response to stimulation, plasma AngⅡ, VP, and ALD concentrations were higher in the offspring exposure to prenatal WD than in the control, associated with an increase of salt intake. The data indicate an increase of risks for cardiovascular diseases such as hypertension in the adult offspring exposed to maternal WD during late gestation.PART 3 Effects of maternal WD during late gestation on expression of proteome in the heart and kidney of the offspringObjective: To determine the expression of proteome in both the heart and kidney in the adult male offspring exposure to maternal WD during late gestation. Methods: Two dimensional electrophoresis (2-DE) was used to detect differences of proteome expression between the control and WD group in the heart and kidney of the adult male offspring.Results: The basic distributions of total proteins were similar in both the heart and kidney in offsprings between the control and WD groups. The numbers of total proteins were 603±25 and 577±32 in the heart between the control and WD groups, in which thirty-three differential protein spots were identified. Total proteins were 975±30 and 1107±42 in the kidney between the control and WD groups, in which thirty-eight differential protein spots were identified.Conclusion: 2-DE profiles with high resolution and reproducibility of total proteins were established in both the heart and kidney between the control and WD groups, and the expressions of differential proteins were found.更多还原