Dong Yafeng, Thompson Loren P
Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Soc Gynecol Investig. 2006 Oct;13(7):483-90. doi: 10.1016/j.jsgi.2006.06.005. Epub 2006 Sep 18.
The purpose of the present study was to quantify the effect of chronic hypoxia on endothelial nitric oxide synthase (eNOS) gene and protein expression of fetal coronary artery segments and cardiac tissue of fetal guinea pig hearts.
Time-mated pregnant guinea pigs (term = 65 days) were housed in room air (NMX, n = 6) or in a hypoxic chamber containing 10.5% O2 for 14 days (HPX14, n = 6). At near term (60 days gestation), fetuses were excised from anesthetized animals via hysterotomy and hearts were removed and weighed. Both coronary artery segments and cardiac ventricle were excised from the same hearts, frozen, and stored at -80 C until ready for study. eNOS mRNA was quantified using real-time polymerase chain reaction (PCR) based on SYBR Green I labeling (BioRad Laboratories, Hercules, CA) using eNOS primers obtained from GeneBank normalized to 18S. eNOS proteins were quantified by Western immunoblotting using eNOS antibody (1:200) and normalized to normoxic controls. eNOS cell-specific localization in the fetal guinea pig heart was performed by double immunofluorescence staining.
Both coronary artery endothelial cells (EC) and cardiomyocytes (CM) but not vascular smooth muscle cells of normoxic hearts exhibited positive immunostaining of eNOS protein. Chronic hypoxia significantly (P < .05) increased both eNOS mRNA and protein levels of coronary artery segments (by 210.6% and 51.4%, respectively) but decreased (P < .05) mRNA and protein of cardiac tissue (by 50.0% and 40.6%, respectively) in the same hearts.
Chronic fetal hypoxia, after 14 days, induces sustained changes in eNOS gene and eNOS protein expression that differ between coronary and cardiac tissue in the fetal guinea pig heart. This study suggests that while the functional roles of altered eNOS expression in hypoxic fetal hearts remain unclear, the site at which eNOS expression is altered may be important in the adaptive response of the fetal heart to hypoxia.
本研究旨在量化慢性缺氧对胎豚鼠心脏冠状动脉段及心脏组织中内皮型一氧化氮合酶（eNOS）基因和蛋白表达的影响。
将同期受孕的豚鼠（孕期65天）饲养于正常空气中（NMX，n = 6）或含10.5%氧气的缺氧舱中14天（HPX14，n = 6）。在妊娠近足月（60天）时，通过子宫切开术从麻醉的动物体内取出胎儿，取出心脏并称重。从同一心脏中取出冠状动脉段和心室，冷冻后储存在-80℃直至准备研究。使用基于SYBR Green I标记的实时聚合酶链反应（PCR）（BioRad Laboratories，Hercules，CA），以从GeneBank获得的eNOS引物并以18S为标准来定量eNOS mRNA。使用eNOS抗体（1:200）通过Western免疫印迹法定量eNOS蛋白，并以常氧对照为标准进行标准化。通过双重免疫荧光染色对胎豚鼠心脏中eNOS进行细胞特异性定位。
常氧心脏的冠状动脉内皮细胞（EC）和心肌细胞（CM）而非血管平滑肌细胞呈现eNOS蛋白阳性免疫染色。慢性缺氧显著（P < .05）增加了冠状动脉段的eNOS mRNA和蛋白水平（分别增加210.6%和51.4%），但同一心脏中，心脏组织的mRNA和蛋白水平降低（P < .05）（分别降低50.0%和40.6%）。
14天的慢性胎儿缺氧可诱导胎豚鼠心脏中冠状动脉和心脏组织的eNOS基因和eNOS蛋白表达持续变化，且两者不同。本研究表明，虽然缺氧胎儿心脏中eNOS表达改变的功能作用尚不清楚，但eNOS表达改变的部位可能在胎儿心脏对缺氧的适应性反应中起重要作用。
