發(fā)布時間：2018-06-03 14:30

  本文選題：孕期肥胖 + 缺血再灌注��； 參考：《廣州醫(yī)科大學》2014年碩士論文

【摘要】：第一部分 孕期肥胖對胎鼠心臟結(jié)構(gòu)和心肌細胞增殖的影響及其機制研究 目的 孕期肥胖通過影響AT1R和AT2R的表達來探討胎鼠心臟結(jié)構(gòu)和心肌細胞增殖改變的機制。 方法 在SD雌鼠妊娠期第1~21天給予高脂飲食,建立妊娠期肥胖動物模型。取21天乳鼠心臟進行原代心肌細胞培養(yǎng)，利用流式細胞技術(shù)檢測心肌細胞的細胞周期。透射電鏡觀察21天胎鼠心臟超微結(jié)構(gòu)的變化；Western-blot法檢測心肌組織AT1R和AT2R蛋白表達的情況； RT-PCR法檢測AT1R和AT2R mRNA的表達情況；染色質(zhì)免疫沉淀方法和GR抗體提取GR-GRE復合物進行RT-PCR擴增檢測GR與GRE結(jié)合力情況。 結(jié)果 1.與對照組相比，妊娠期第1天，肥胖組的孕鼠體重無顯著性差異，但在妊娠期第21天，肥胖組的孕鼠體重顯著增加（P0.05）。 2.肥胖組和對照組胎鼠的心臟重量與體重沒有統(tǒng)計學差異，但肥胖組胎鼠心臟體重的比值明顯高于對照組(P0.05)。 3.與對照組相比，肥胖組胎鼠心肌細胞在G1期細胞數(shù)量明顯增加，而S期和G2期，心肌細胞數(shù)量顯著減少（P0.05）。 4.孕期肥胖改變胎鼠心臟超微結(jié)構(gòu)，包括肌原纖維排列紊亂，微絲模糊，變小，甚至消失；線粒體腫脹，線粒體嵴部分或全部消失，線粒體呈空泡化。 5. Western-blot結(jié)果顯示，與對照組相比，肥胖組胎鼠心臟AT1R蛋白表達水平明顯降低，而AT2R蛋白水平顯著升高（P0.05）。 6.RT-PCR結(jié)果顯示，與對照組相比，肥胖組胎鼠心臟AT1aR mRNA水平明顯降低；而AT2R mRNA水平顯著升高（P0.05）。 7. Western-blot結(jié)果顯示，與對照組相比，肥胖組胎鼠心臟GR總蛋白、GR核蛋白和GR mRNA水平顯著降低（P0.05）。 8. ChIP結(jié)果顯示在孕期肥胖減少AT1R和AT2R啟動子上GRE與GR的結(jié)合。 小結(jié) 1.孕期肥胖導致胎鼠心臟超微結(jié)構(gòu)（包括肌原纖維紊亂、線粒體腫脹、線粒體嵴消失甚至空泡化）的改變和心肌細胞增殖的抑制，這可能與血管緊張素Ⅱ受體的改變有關(guān)。 2.孕期肥胖降低胎鼠心臟AT1aR和AT2R啟動子上GR與GRE的結(jié)合，導致胎鼠心臟血管緊張素Ⅱ受體的改變。 第二部分 孕期肥胖對成年子代心功能異常程序化的影響及其機制研究 目的 孕期肥胖通過影響AT1R和AT2R的表達以及對子代大鼠心肌缺血再灌注損傷來探討孕期肥胖導致成年子代鼠心臟功能異常的機制，并且確定是否存在性別差異。 方法 在SD雌鼠妊娠期第1~21天給予高脂飲食,建立妊娠期肥胖動物模型。出生后，，肥胖組和對照組母鼠正常飲食，子代大鼠同等條件下飼養(yǎng)至3個月齡作為實驗用動物。小動物超聲成像系統(tǒng)檢測成年子代大鼠心臟功能；子代大鼠心臟建立langendroff系統(tǒng)檢測大鼠心臟缺血再灌注損傷的敏感性；TTC法測定子代大鼠缺血再灌注后心臟梗死面積；LDH試劑盒測定灌流液中LDH的活性；EIA法測定子代大鼠血清Ang II含量；Western blot法檢測子代大鼠心肌組織AT1R和AT2R蛋白表達的情況；RT-PCR法檢測AT1R和AT2R mRNA的表達；染色質(zhì)免疫沉淀方法和GR抗體提取GR-GRE復合物進行RT-PCR擴增檢測GR與GRE結(jié)合力情況。 結(jié)果 1.結(jié)果顯示與對照組相比，肥胖組子代雄性大鼠的體重沒有變化，而心臟重量和心臟體重比值顯著升高（P0.05），而雌性大鼠體重、心臟重量和心臟體重比值均沒有改變。 2.超聲結(jié)果顯示在短軸M模式下測量子代大鼠收縮功能，與對照組相比，肥胖組子代雄性大鼠的左心室舒張期前壁厚度（LVAW;d）、左心室收縮期前壁厚度（LVAW;s）、左心室舒張期后壁厚度（LVPW;d）、左心室收縮期后壁厚度（LVPW;s）、左心室射血分數(shù)EF（%）和左心室短軸縮短速率FS（%）均顯著增加，而左室舒張末期內(nèi)徑（LVID;d）、左室收縮末期內(nèi)徑（LVID;s）明顯減少（P0.05）。而肥胖組子代雌性大鼠室壁厚度和心室內(nèi)徑均無顯著差異。 3.在二尖瓣灌流測量模式下測量子代大鼠舒張功能，肥胖組子代雄性大鼠和雌性大鼠的舒張功能均沒有改變。測量的指標有MVA, MVE, E/A ratio, DT,IVRT, and ET。 4.孕期肥胖加重子代雄性大鼠心臟缺血再灌注心功能的損傷、梗死面積和灌流液中LDH的活性（P0.05）。而子代雌性大鼠心臟則沒有改變。 5.孕期肥胖增加子代雄性大鼠血清中AngII的含量（P0.05），而雌性大鼠則沒有變化。孕期肥胖增加子代雄性大鼠心臟AT2R蛋白和mRNA的表達（P0.05），而雌性大鼠心臟則沒有改變。然而，孕期肥胖對子代雄性和雌性大鼠心臟AT1R的表達沒有任何影響。 6.拮抗AT2R的表達可以逆轉(zhuǎn)孕期肥胖增加子代雄性大鼠心肌缺血再灌注損傷敏感性。 7.孕期肥胖降低子代雄性大鼠心臟AT2R啟動子上GR與GRE的結(jié)合，這是由于雄性大鼠心臟GR核蛋白量的減少和結(jié)合力的降低導致的。 小結(jié) 1.孕期肥胖沒有改變子代心臟收縮和舒張功能，但發(fā)現(xiàn)子代有心肌肥厚的傾向，并且存在性別差異。 2.孕期肥胖通過下調(diào)GR的表達來增加AT2R基因的表達，從而增加子代缺血再灌注損傷的敏感性，并且存在性別差異。 全文結(jié)論 本研究采用妊娠期肥胖動物模型探討了血管緊張素II受體在孕期肥胖對子代心功能影響中的作用。孕期肥胖改變胎鼠心臟結(jié)構(gòu)和抑制心肌細胞增殖，其機制可能與AT2R/AT1R的比值改變有關(guān)；孕期肥胖通過下調(diào)子代心臟GR的表達來增加AT2R基因的表達，從而加重子代心肌缺血再灌注損傷，并且存在性別差異。
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.256

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