宫内宫外营养干预对成年期肺血管功能的影响及表观调控机制探索

发布时间：2019-05-27 12:42

【摘要】：目的：流行病学调查和实验室研究均已发现胎儿期和婴幼儿时期的营养不良会导致成年期肺血管重塑和肺动脉高压的发病风险增加。这两个时期的营养状况会分别影响成年期疾病的发生发展。本研究旨在通过建立宫内营养受限和宫内及哺乳期均营养限制的雄性SD大鼠模型,探讨宫内生长迟缓(intrauterine growth retardation, IUGR)子代在哺乳期继续限制饮食能否降低其在成年期发生肺血管重塑和肺动脉高压的风险,并以此进一步研究肺动脉相关基因的表达改变及其潜在的表观调控机制。方法： 1.模型建立：雌雄SD大鼠交配,怀孕当天将雌鼠随机分成限食组和正常进食组。限食组母鼠整个孕期和哺乳期内的每日进食量为正常组的50%。正常进食组母鼠所生子鼠由其本身继续喂养为对照组；孕期限食组母鼠所生子鼠部分由正常进食组母鼠喂养为IUGR组,部分继续由限食组母鼠喂养为生前生后生长迟缓(intrauterine and postnatal growth retardation, IPGR)组。仅保留雄性SD大鼠做后续研究。 2.14周成年雄性SD大鼠,插管测量右心室平均压和肺动脉平均压。取离体心脏,分别称量右心室、左心室加室间隔的重量,计算右心肥厚指数。取左肺作病理切片,免疫组织化学染色观察肺血管肌化程度。 3.荧光定量PCR检测肺组织中eNOS基因的表达水平。 4.染色质免疫共沉淀分析基因启动子区域的组蛋白结合情况。结果： 1.体重监测： 1)SD雄性大鼠的出生体重：母亲限食(mother food restricted, MFR)组明显轻于对照组。 2)SD雄性大鼠在生后21天和14周体重：对照组、IUGR组和IPGR组三组体重分布具有明显差异,各时间点两两比较也均有明显差异,呈对照组IUGR组IPGR组。 2.14周SD雄性大鼠肺动脉平均压：IUGR组相比对照组有所升高；IPGR组相比IUGR组出现一定程度的降低。 3. IUGR组肺小动脉中膜厚度相比对照组增厚；IPGR组相比IUGR组肺小动脉中膜厚度有所减小。 4.14周IUGR组大鼠肺组织中eNOS表达量转录水平上相比对照组升高,IPGR组相比IUGR组出现一定程度的下降。 5. eNOS基因启动子区域组蛋白修饰情况：IUGR组和IPGR组的eNOS启动子区域所结合组蛋白H3K9met3量相比对照组升高。结论： IUGR雄性SD大鼠在成年期肺动脉压力升高,肺血管平滑肌增厚。而IUGR经过哺乳期饮食限制后,成年期发生肺血管功能失调的风险有所下降。IUGR子代的肺组织中eNOS基因表达量的改变可能在此肺血管重塑过程中起到一定作用,其表观调控机制目前仍需进一步的探索。
[Abstract]:Objective: epidemiological and laboratory studies have found that malnutrition in fetal and infant stages can lead to increased risk of pulmonary vascular remodeling and pulmonary hypertension in adulthood. The nutritional status of these two periods will affect the occurrence and development of adult diseases. The purpose of this study was to establish a male SD rat model with intrauterine nutrition restriction and both intrauterine and lactating nutrition restriction, and to explore the (intrauterine growth retardation, of intrauterine growth retardation. IUGR) whether diet restriction during lactation can reduce the risk of pulmonary vascular remodeling and pulmonary hypertension in adulthood, and further study the changes of pulmonary artery related gene expression and its potential apparent regulatory mechanism. Method: 1. The model was established: male and female SD rats mated and were randomly divided into food restriction group and normal eating group on the day of pregnancy. The daily intake of female rats in the restricted diet group was 50% of that in the normal group during the whole pregnancy and lactation period. The offspring of the female rats in the normal eating group were fed continuously from themselves to the control group. The offspring of the pregnant group were partly fed by the normal eating group to the IUGR group, and some of the female mice were fed to the postmortem growth retardation (intrauterine and postnatal growth retardation, IPGR) group by the feeding restriction group. Only male SD rats were retained for follow-up study. 2.14 weeks adult male SD rats were intubated to measure the mean pressure of right ventricle and pulmonary artery. The right ventricular hypertrophy index was calculated by weighing the weight of right ventricle and left ventricle plus interventricular septum. The left lung was taken for pathological section and the degree of pulmonary vascular muscle was observed by immunohistochemical staining. 3. The expression of eNOS gene in lung tissue was detected by fluorescence quantitative PCR. 4. The histone binding in the promoter region of the gene was analyzed by chromatin immunoprecipitation. Result: 1. Weight monitoring: 1) birth weight of SD male rats: the birth weight of (mother food restricted, MFR) group was significantly lighter than that of control group. 2) body weight of male SD rats at 21 and 14 weeks after birth: there were significant differences in body weight distribution between control group, IUGR group and IPGR group, and there were also significant differences at each time point, showing control group IUGR group IPGR group. 2.14 weeks after SD, the mean pulmonary artery pressure of male SD rats was higher than that of the control group, and the mean pulmonary artery pressure of the IPGR group was lower than that of the control group to a certain extent. 3. The intima thickness of pulmonary arterioles in IUGR group was thicker than that in control group, and that in IPGR group was lower than that in IUGR group. 4. At the 14th week, the transcriptional level of eNOS expression in lung tissue of IUGR group was higher than that of control group, and that of IPGR group was lower than that of IUGR group to a certain extent. 5. Histone modification of eNOS gene promoter region: the amount of histone H _ 3K9met3 bound to eNOS promoter region in IUGR group and IPGR group was higher than that in control group. Conclusion: the pulmonary artery pressure and pulmonary vascular smooth muscle of IUGR male SD rats are increased in adult. However, after lactation diet restriction, the risk of pulmonary vascular dysfunction in adulthood decreased. The change of eNOS gene expression in lung tissue of IUGR offspring may play a role in the process of pulmonary vascular remodeling. At present, its apparent regulation mechanism still needs to be further explored.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R714.5

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