Aptosis in an ovine model of intrauterine growth restriction (IUGR)

描述（由申请人提供）：宫内生长受限（IUGR）是婴儿死亡率和发病率增加的重要原因。在IUGR期间，已经描述了许多胎盘异常，包括滋养细胞凋亡的增加。滋养细胞异常凋亡可能导致氧和营养交换受损，提示细胞凋亡在IUGR的发展中起作用。为了更好地了解IUGR妊娠中的细胞凋亡，我们将利用已建立的热疗绵羊IUGR模型，该模型相当模仿人类疾病。我们建议观察绵羊妊娠早期、高温20天、胎盘生长高峰55天和胎儿生长高峰80天的胎盘细胞凋亡情况。我们的长期目标是：1)确定高温诱导绵羊PI-IUGR模型中胎盘的体内凋亡效应、DNA降解、增殖率、端粒酶活性和细胞角蛋白18切割；2)阐明绵羊P1-1UGR胎盘中抗凋亡分子BCL-2和XIAP以及促凋亡分子BAX、caspase 3和caspase 9的表达。探讨热疗诱导PI-IUGR细胞凋亡过程中HSP27与HIF-2a的相关性及表达情况。3)探讨热疗、缺氧或两者均对正常绵羊胎盘培养双核细胞（BNC）凋亡、细胞角蛋白18切割、端粒酶活性及BCL- 2、XIAP、BAX、caspase 3、9、HSP27、HIF-2a表达的影响。为了完成这些，我们将使用末端脱氧核苷酸转移酶介导的dUTP镍端标记技术（TUNEL）和细胞角蛋白18切割表达来确定细胞凋亡。此外，我们将使用端粒酶重复扩增协议（TRAP）技术确定与我们的IUGR模型相关的端粒酶活性。为了进一步研究细胞凋亡，我们将测定经高温处理的动物胎盘中Bcl-2、Bax、caspase 3和9的蛋白激活水平，并与对照组进行比较。此外，我们将确定热疗对治疗动物胎盘中XIAP和HIF- 2a表达的影响。为了在体外研究细胞凋亡，我们将确定高热和缺氧对培养的胎盘bnc的影响。

DESCRIPTION (provided by applicant): Intrauterine growth restriction (IUGR) is a significant cause of increased infant mortality and morbidity. A number of placental abnormalities have been described during IUGR including an increase in trophoblast apoptosis. Abnormal trophoblast apoptosis may lead to impaired oxygen and nutrient exchange, suggesting a role for apoptosis in the development of IUGR. To better understand apoptosis in IUGR pregnancies we will utilize an established hyperthermia sheep IUGR model which mimics the human disease considerably. We propose to look at apoptosis in the sheep placentae early in gestation and in sheep exposed to hyperthermia for 20 days, 55 days when placental growth is at its peak and 80 days near term when fetal growth is maximal. Our long-term goals are: 1) to determine the in-vivo placental apoptotic effects, DNA degradation, proliferation rate, telomerase activity and cytokeratin 18 cleavage in our model of hyperthermia induced PI-IUGR in the sheep, 2) to elucidate the expression anti-apoptotic molecules BCL-2 and XIAP and the pro-apoptotic molecules BAX, caspase 3 and caspase 9 in the sheep P1-1UGR placentae. To determine the correlation and expression of HSP27 and of HIF-2a during apoptosis in hyperthermia induced PI-IUGR, and 3) to determine the effect of hyperthermia, hypoxia or both on apoptosis, cytokeratin 18 cleavage, telomerase activity and expression of BCL- 2, XIAP, BAX, caspase 3 and 9, HSP27 and HIF-2a in cultured placental binucleated cells (BNC) obtained from normal sheep placentomes. In order to accomplish these we will determine apoptosis using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique (TUNEL) and cytokeratin 18cleavage expression. Also we will determine the telomerase activity associated with our model of IUGR using the Telomerase Repeat Amplification Protocol (TRAP) technique. To further study apoptosis, we will determine the protein levels of Bcl-2, Bax, and caspase 3 and 9 activation in placentae from animals treated with hyperthermia as compared to controls. Also, we will determine the effects of hyperthermia in the expression of XIAP and HIF- 2a in the placentae from treated animals. To study apoptosis in-vitro, we will determine the effects of hyperthermia and hypoxia on cultured placental BNCs. PUBLIC HEALTH RELEVANCE (See instructions): These studies will help elucidate the in-vivo and in-vitro apoptotic effects of hyperthermia in the development of PI-IUGR in the sheep and will provide insights for placental dysfunction and growth delay associated with compromised pregnancies like IUGR.