孕期高脂膳食对子代肝脏线粒体编程的影响及机制

It is suggested that mitochondria might be the putative targets for the fetal programming of adult disease. We all found that high fat diet during gestation induced oxidative stress, lipid metabolism disorder and mitochondral damage deduced from the results of microarry and bioinformatics analysis. So, this project aims to test the hypothesis that high lipid diet during gestation programed the mitochondrial develpoment by induring oxidative stress and disording insulin signalling pathway. Pregnant women are recruited and serum lipid, insulin, glucose levels were measured and the relation between the levels of these biomarker to dietary fat intake during gestation were analized. Wistar rats were fed with high fat diet during gestation or befor gestation, the serum or/and liver were sampled to measure insuin, lipid, glucose levels and oxidative stress. Liver ATP content, oxidative stress, TCA and ETL enzemy activities were measured. In order to observe the effects of oxidative stress and high lipid on mitochondria directly, HepG2 cell line was treated with glucoseoxidase or free fatty acids to make oxidative or insulin resistant cell model. Mitochondria membrane, oxidative state, ETL enzyme activity were meaured. If the hypothseis is tested to be right, the mechanism under the fetal programing will be elucified.

研究发现，孕期膳食可能影响子代线粒体的功能，本课题组的前期研究结果提示孕期高脂膳食可能通过氧化损伤和胰岛素信号损伤肝脏线粒体。为此，我们拟在人群、动物和细胞三个层面，采用基因芯片和生物信息学、电子显微镜、激光共聚焦显微镜、分子杂交、蛋白质双向电泳等技术，分析1）孕妇外周血、胎盘、脐带血的胰岛素、氧化应激标志物和线粒体形态及功能、线粒体膜流动性和膜电位，2）孕期进食高脂饲料大鼠的子代（新生、断乳和成年鼠）肝脏线粒体的形态结构、功能及蛋白质组谱、肝脏基因表达谱、胰岛素信号和氧化应激标志分子的活性或表达，3）体外氧化应激和胰岛素抵抗细胞模型的线粒体形态结构和功能的改变以及胰岛素信号通路、氧化应激标志物和线粒体合成过程关键基因的表达；验证"孕期高脂膳食通过胰岛素信号和氧化应激改变子代肝脏线粒体编程"这一假设，解释子代健康受孕期膳食影响的机制，为相关疾病的预防提供新的思路和策略。

动物研究发现生命早期母亲的高能量膳食或者能量限制膳食均可编程子代的超重肥胖，增加子代成年后慢性非传染性疾病的发病风险。超重/肥胖是能量摄取和消耗不平衡所导致的，能量代谢的中心时线粒体，因此，本研究提出妊娠期高脂高能量膳食可能编程子代肝脏的线粒体，使得线粒体的数量和功能发生损伤。通过本项目的研究，结果发现以孕期高脂血症/高血糖可导致子代脐带血线粒体数量的减少、降低胎盘胆固醇的转运，但对脐带血的氧化应激和胰岛素水平以及线粒体功能的影响不大；说明胎儿发育对母亲的高脂高能量膳食有较好的适应性；动物研究发现，孕期高脂饲料喂养子代肝脏线粒体呼吸酶系统相关基因、ATP生成基因等大多表达出现先升高后降低的变化趋势，同时虽然线粒体拷贝数降低，但对ATP的含量、酶的活性这些能够体现线粒体功能的指标的影响较小，提示线粒体编程过程对宫内高脂高能量暴露的适应；胰岛素信号通路中的关键因子如LXR、PPARγ等的基因在肝脏和胎盘的表达受孕期血脂水平的影响，但脐带血和子代成年动物血中这些指标的变化无显著性差异，氧化应激水平也未发现有显著变化，这些结果提示，孕期高脂高能量膳食/高脂血症对子代肝脏能量代谢和线粒体编程具有影响，但仅限于基因层面，在正常情况下，线粒体功能未有显著改变。