孕期肥胖对子代心功能异常程序化的影响及其机制研究

发布时间：2018-06-03 14:30

本文选题：孕期肥胖 + 缺血再灌注　；参考：《广州医科大学》2014年硕士论文

【摘要】：第一部分孕期肥胖对胎鼠心脏结构和心肌细胞增殖的影响及其机制研究目的孕期肥胖通过影响AT1R和AT2R的表达来探讨胎鼠心脏结构和心肌细胞增殖改变的机制。方法在SD雌鼠妊娠期第1~21天给予高脂饮食,建立妊娠期肥胖动物模型。取21天乳鼠心脏进行原代心肌细胞培养，利用流式细胞技术检测心肌细胞的细胞周期。透射电镜观察21天胎鼠心脏超微结构的变化；Western-blot法检测心肌组织AT1R和AT2R蛋白表达的情况； RT-PCR法检测AT1R和AT2R mRNA的表达情况；染色质免疫沉淀方法和GR抗体提取GR-GRE复合物进行RT-PCR扩增检测GR与GRE结合力情况。结果 1.与对照组相比，妊娠期第1天，肥胖组的孕鼠体重无显著性差异，但在妊娠期第21天，肥胖组的孕鼠体重显著增加（P0.05）。 2.肥胖组和对照组胎鼠的心脏重量与体重没有统计学差异，但肥胖组胎鼠心脏体重的比值明显高于对照组(P0.05)。 3.与对照组相比，肥胖组胎鼠心肌细胞在G1期细胞数量明显增加，而S期和G2期，心肌细胞数量显著减少（P0.05）。 4.孕期肥胖改变胎鼠心脏超微结构，包括肌原纤维排列紊乱，微丝模糊，变小，甚至消失；线粒体肿胀，线粒体嵴部分或全部消失，线粒体呈空泡化。 5. Western-blot结果显示，与对照组相比，肥胖组胎鼠心脏AT1R蛋白表达水平明显降低，而AT2R蛋白水平显著升高（P0.05）。 6.RT-PCR结果显示，与对照组相比，肥胖组胎鼠心脏AT1aR mRNA水平明显降低；而AT2R mRNA水平显著升高（P0.05）。 7. Western-blot结果显示，与对照组相比，肥胖组胎鼠心脏GR总蛋白、GR核蛋白和GR mRNA水平显著降低（P0.05）。 8. ChIP结果显示在孕期肥胖减少AT1R和AT2R启动子上GRE与GR的结合。小结 1.孕期肥胖导致胎鼠心脏超微结构（包括肌原纤维紊乱、线粒体肿胀、线粒体嵴消失甚至空泡化）的改变和心肌细胞增殖的抑制，这可能与血管紧张素Ⅱ受体的改变有关。 2.孕期肥胖降低胎鼠心脏AT1aR和AT2R启动子上GR与GRE的结合，导致胎鼠心脏血管紧张素Ⅱ受体的改变。第二部分孕期肥胖对成年子代心功能异常程序化的影响及其机制研究目的孕期肥胖通过影响AT1R和AT2R的表达以及对子代大鼠心肌缺血再灌注损伤来探讨孕期肥胖导致成年子代鼠心脏功能异常的机制，并且确定是否存在性别差异。方法在SD雌鼠妊娠期第1~21天给予高脂饮食,建立妊娠期肥胖动物模型。出生后，，肥胖组和对照组母鼠正常饮食，子代大鼠同等条件下饲养至3个月龄作为实验用动物。小动物超声成像系统检测成年子代大鼠心脏功能；子代大鼠心脏建立langendroff系统检测大鼠心脏缺血再灌注损伤的敏感性；TTC法测定子代大鼠缺血再灌注后心脏梗死面积；LDH试剂盒测定灌流液中LDH的活性；EIA法测定子代大鼠血清Ang II含量；Western blot法检测子代大鼠心肌组织AT1R和AT2R蛋白表达的情况；RT-PCR法检测AT1R和AT2R mRNA的表达；染色质免疫沉淀方法和GR抗体提取GR-GRE复合物进行RT-PCR扩增检测GR与GRE结合力情况。结果 1.结果显示与对照组相比，肥胖组子代雄性大鼠的体重没有变化，而心脏重量和心脏体重比值显著升高（P0.05），而雌性大鼠体重、心脏重量和心脏体重比值均没有改变。 2.超声结果显示在短轴M模式下测量子代大鼠收缩功能，与对照组相比，肥胖组子代雄性大鼠的左心室舒张期前壁厚度（LVAW;d）、左心室收缩期前壁厚度（LVAW;s）、左心室舒张期后壁厚度（LVPW;d）、左心室收缩期后壁厚度（LVPW;s）、左心室射血分数EF（%）和左心室短轴缩短速率FS（%）均显著增加，而左室舒张末期内径（LVID;d）、左室收缩末期内径（LVID;s）明显减少（P0.05）。而肥胖组子代雌性大鼠室壁厚度和心室内径均无显著差异。 3.在二尖瓣灌流测量模式下测量子代大鼠舒张功能，肥胖组子代雄性大鼠和雌性大鼠的舒张功能均没有改变。测量的指标有MVA, MVE, E/A ratio, DT,IVRT, and ET。 4.孕期肥胖加重子代雄性大鼠心脏缺血再灌注心功能的损伤、梗死面积和灌流液中LDH的活性（P0.05）。而子代雌性大鼠心脏则没有改变。 5.孕期肥胖增加子代雄性大鼠血清中AngII的含量（P0.05），而雌性大鼠则没有变化。孕期肥胖增加子代雄性大鼠心脏AT2R蛋白和mRNA的表达（P0.05），而雌性大鼠心脏则没有改变。然而，孕期肥胖对子代雄性和雌性大鼠心脏AT1R的表达没有任何影响。 6.拮抗AT2R的表达可以逆转孕期肥胖增加子代雄性大鼠心肌缺血再灌注损伤敏感性。 7.孕期肥胖降低子代雄性大鼠心脏AT2R启动子上GR与GRE的结合，这是由于雄性大鼠心脏GR核蛋白量的减少和结合力的降低导致的。小结 1.孕期肥胖没有改变子代心脏收缩和舒张功能，但发现子代有心肌肥厚的倾向，并且存在性别差异。 2.孕期肥胖通过下调GR的表达来增加AT2R基因的表达，从而增加子代缺血再灌注损伤的敏感性，并且存在性别差异。全文结论本研究采用妊娠期肥胖动物模型探讨了血管紧张素II受体在孕期肥胖对子代心功能影响中的作用。孕期肥胖改变胎鼠心脏结构和抑制心肌细胞增殖，其机制可能与AT2R/AT1R的比值改变有关；孕期肥胖通过下调子代心脏GR的表达来增加AT2R基因的表达，从而加重子代心肌缺血再灌注损伤，并且存在性别差异。
[Abstract]:Part one
Effect of gestational obesity on cardiac structure and cardiomyocyte proliferation in fetal rats and its mechanism
objective
The mechanism of obesity during pregnancy is to explore the mechanism of fetal heart structure and cardiomyocyte proliferation by influencing the expression of AT1R and AT2R.
Method
The high fat diet was given to the SD female rats on the 1~21 day of pregnancy, and the obese animal model of pregnancy was established. The heart of the rat was cultured for 21 days, and the cell cycle of the cardiac myocytes was detected by flow cytometry. The ultrastructure of the heart was observed by transmission electron microscope for 21 days, and the Western-blot method was used to detect the AT1R and AT2R eggs of the myocardium. The expression of white expression, the expression of AT1R and AT2R mRNA were detected by RT-PCR, and the binding force of GR and GRE was detected by RT-PCR amplification by RT-PCR amplification by chromatin immunoprecipitation and GR antibody extraction of GR-GRE.
Result
1. compared with the control group, there was no significant difference in body weight between the obese group and the control group on the first day of gestation, but in the twenty-first days of gestation, the weight of the pregnant rats in the obese group increased significantly (P0.05).
2. there was no significant difference in heart weight and body weight between the obese group and the control group, but the body weight ratio of the obese rats was significantly higher than that of the control group (P0.05).
3. compared with the control group, the number of cardiomyocytes in the G1 phase increased significantly in the obese group, while in the S and G2 phases, the number of cardiomyocytes decreased significantly (P0.05).
4. pregnancy obesity changed the ultrastructure of fetal rat heart, including myofibrils disorder, microfilament blurred, small and even disappeared, mitochondria swelled, mitochondrial crista disappeared and mitochondria were vacuolated.
5. Western-blot results showed that compared with the control group, the expression of AT1R protein in the heart of obese rats decreased significantly, while the level of AT2R protein increased significantly (P0.05).
6.RT-PCR results showed that compared with the control group, the AT1aR mRNA level in the heart of the obese group was significantly decreased, while the AT2R mRNA level increased significantly (P0.05).
7. Western-blot results showed that the total GR protein, GR nucleoprotein and GR mRNA levels in the heart of obese rats were significantly lower than those in the control group (P0.05).
8. ChIP results showed that obesity during pregnancy reduced the combination of GRE and GR on AT1R and AT2R promoters.
Summary
1. pregnancy obesity induced fetal rat cardiac ultrastructure (including myofibrillar disorder, mitochondrial swelling, mitochondrial crista disappearance and vacuolization) and the inhibition of myocardial cell proliferation, which may be associated with changes in angiotensin II receptor.
Obesity during pregnancy reduced the binding of GR to GRE on the AT1aR and AT2R promoter of fetal rat heart, resulting in the change of angiotensin II receptor in fetal rat heart. 2.
The second part
Effect of gestational obesity on programmed dysfunction of cardiac function in adult offspring and its mechanism
objective
The effect of obesity on the expression of AT1R and AT2R and the myocardial ischemia reperfusion injury in the offspring of the offspring to explore the mechanism of abnormal cardiac function in adult offspring rats during pregnancy and determine whether there is a gender difference.
Method
A high fat diet was given to a high fat diet in SD female rats on the 1~21 day of pregnancy. After birth, the obese group and the control group had normal diet. The offspring of the offspring were fed to 3 months of age as experimental animals. The ultrasonic imaging system of small animals was used to detect the cardiac function of adult offspring rats; the heart of the offspring rat was set up to establish langendr. Off system was used to detect the sensitivity of myocardial ischemia reperfusion injury in rats; TTC assay was used to determine the infarct area of the rat after ischemia and reperfusion; the activity of LDH in the perfusion fluid was measured by LDH kit; the content of Ang II in the serum of the offspring rats was measured by EIA; the Western blot method was used to detect the expression of AT1R and AT2R protein in the myocardium of the offspring rats; PCR method was used to detect the expression of AT1R and AT2R mRNA. Chromatin immunoprecipitation method and GR antibody were used to extract GR-GRE complex. RT-PCR amplification was used to detect the binding force between GR and GRE.
Result
1. the results showed that compared with the control group, the weight of the male rats in the obese group did not change, but the heart weight and the heart weight ratio increased significantly (P0.05), while the weight of the female rats, the heart weight and the ratio of the heart weight were not changed.
2. ultrasound results showed that the systolic function of the quantum generation rats was measured in the short axis M mode. Compared with the control group, the left ventricular diastolic anterior wall thickness (LVAW; d), the anterior wall thickness of left ventricular systole (LVAW; s), the left ventricular diastolic wall thickness (LVPW; D), the left ventricular posterior wall thickness (LVPW; s), and the left ventricular ejection fraction EF (d) were compared with the control group. The short axis shortening rate of left ventricular (%) and FS (%) increased significantly, while the left ventricular end diastolic diameter (LVID; d) and left ventricular end systolic diameter (LVID; s) decreased significantly (P0.05), but there was no significant difference in the ventricular wall thickness and ventricular diameter in the obese group of female rats.
3. the diastolic function of the quantum generation rats was measured under the mitral valve perfusion model. The diastolic function of the male and female rats in the obese group did not change. The measurements were MVA, MVE, E/A ratio, DT, IVRT, and ET..
In 4. obese and obese rats, the cardiac function damage, infarct area and LDH activity in the perfusion fluid (P0.05) were found in obese and baryon male rats, while the heart of the female offspring of the offspring did not change.
5. pregnancy obesity increased the content of AngII in the serum of the offspring male rats (P0.05), but the female rats did not change. Pregnancy obesity increased the expression of AT2R protein and mRNA in the heart of the offspring male rats (P0.05), but the heart of the female rats did not change. However, the pregnancy obesity had no effect on the expression of AT1R in the male and female rats.
6. antagonizing the expression of AT2R can reverse obesity during pregnancy and increase the sensitivity of offspring rats to myocardial ischemia-reperfusion injury.
7. pregnancy obesity reduces the combination of GR and GRE on the cardiac AT2R promoter of the male rats, which is due to the decrease of the cardiac GR nucleoprotein in the male rat heart and the decrease of the binding power.
Summary
Obesity during pregnancy did not change the systolic and diastolic function of the offspring, but it was found that the offspring had the tendency of cardiac hypertrophy, and there was gender difference between the 1. groups.
In the 2. trimester, obesity increased the expression of AT2R gene by down regulating the expression of GR, thereby increasing the sensitivity of offspring to ischemia-reperfusion injury, and there was gender difference.
Full text conclusion
In this study, the effect of angiotensin II receptor on the cardiac function of the offspring during pregnancy was investigated using an animal model of pregnancy obesity. Obesity during pregnancy changes the heart structure and inhibits the proliferation of cardiac myocytes in pregnant rats. The mechanism may be related to the change in the ratio of AT2R/AT1R, and obesity through the lower modulation of the heart GR is increased by AT during pregnancy. The expression of 2R gene is associated with myocardial ischemia reperfusion injury, and there are gender differences.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.256

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