脂蛋白脂酶基因在子痫前期胎盘中的变化及意义

目的研究脂蛋白脂酶(lipoproteinlipase,LPL)mRNA在于痫前期患者胎盘组织中的表达与定位,探讨其在子痫前期病理生理过程中的作用。方法利用cDNA表达谱芯片检查子痫前期胎盘组织与正常胎盘组织之间的差异表达基因;根据筛选结果,采用半定量RT-PCR检测子痫前期患者胎盘组织(研究组)和正常孕妇胎盘组织(对照组)中LPLmRNA的表达;以原位杂交方法进行定位。结果在4轮杂交过程中,共筛选出22条有差异表达的基因,其中LPL基因为表达降低基因之一;正常胎盘组织和子痫前期胎盘组织中均存在LPLmRNA,子痫前期胎盘组织中LPLmRNA表达明显低于正常胎盘组织(0.208±0.067vs0.524±0.139,P<0.05);LPLmRNA分布在胎盘绒毛滋养细胞胞浆。结论胎盘组织中LPL的低表达可能参与子痫前期的发病过程。     

Decreased expression of the angiogenic regulators CYR61 (CCN1) and NOV (CCN3) in human placenta is associated with pre-eclampsia

The pregnancy disorder pre-eclampsia (PE) is thought to be caused in part by shallow invasion of the extravillous trophoblast (EVT) leading to uteroplacental insufficiency and hypoxia. Here, we focused on the expressions of cysteine-rich 61 (CYR61, CCN1) and nephroblastoma overexpressed (NOV, CCN3), members of the CCN family of angiogenic regulators, in human placenta during normal pregnancy compared with pre-eclamptic and HELLP placentae using quantitative RT-PCR, western blotting and immunocytochemistry. During normal pregnancy, both proteins showed increasing expression levels and were strongly coexpressed in endothelial cells of vessels, stromal cells and interstitial EVT giant cells. However, NOV showed an earlier onset of expression in villous endothelial cells during gestation compared with CYR61, which may signify distinct roles of these proteins in placental angiogenesis. In early-onset pre-eclamptic placentae, both CYR61 and NOV were expressed at a significantly lower level compared with normal matched controls. This decrease of CYR61 and NOV in pre-eclamptic placentae is not associated with a decrease of the endothelial marker CD34 or vimentin. No obvious changes in the localization of CYR61 and NOV in pre-eclamptic placentae were detected but a change in the intracellular distribution in trophoblast giant cells. Our data point to a potential role of both molecules in the pathogenesis of early-onset PE.     

Lack of human leukocyte antigen-G expression in extravillous trophoblasts is associated with pre-eclampsia

Pre-eclampsia, a common complication of first pregnancies, is thought to result from a poorly perfused placenta and may reflect an abnormal maternal immune reaction to the hemiallogenic fetus. Human leukocyte antigen (HLA)-G, a major histocompatibility tissue-specific antigen expressed in extravillous trophoblast cells (fetal-derived), may protect trophoblasts from maternal-fetal immune intolerance and allow these cells to invade the uterus. Through RNA in-situ hybridization analysis, we studied the expression pattern of HLA-G in normal placentae and placentae from pregnancies complicated by severe pre-eclampsia. In normal placenta we found HLA-G expression in the anchoring extravillous trophoblasts with an increasing gradient of expression in the more invasive cells. However, in nine out of 10 pre-eclamptic placentae HLA-G expression was absent or reduced. We conclude that HLA-G is normally expressed in invasive trophoblasts and HLA-G expression is defective in most pre-eclamptic placentae. We propose that trophoblasts lacking HLA-G are vulnerable to attack by the maternal immune system. These defective trophoblasts will be unable to invade the maternal spiral arteries effectively, thereby developing vessels which cannot adequately nourish the developing placenta. This poorly perfused placenta may initiate the systemic cascade of events associated with pre-eclampsia.     

Expression profile of trophoblast invasion-associated genes in the pre-eclamptic placenta

The primary pathology of pre-eclampsia is thought be a defect in placentation due to failure of trophoblast invasion. Here, we aim to identify the expression profile of invasion-associated genes in the pre-eclamptic placenta. Messenger RNA (mRNA) expression levels of extracellular matrix molecule-related genes in five pre-eclamptic placentas and in five strictly matched normal placentas were assayed using complementary DNA (cDNA) microarrays representing over 220 human cytokine-associated or hormone-associated genes. Results demonstrated greater than two-fold higher expression of 18 extracellular matrix molecule genes, including cadherin, collagen, integrin and selectin, in the pre-eclamptic placenta. Extracellular matrix molecule degradation-related genes, including matrix metalloproteinase (MMP)-10, MMP-13, MMP-15, tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-3, plasminogen and plasminogen activator, were also highly expressed in the pre-eclamptic placenta, compared to the normal placenta. Results suggest that the abnormal expression profiles of extracellular matrix molecules and degrading proteinases might be associated with the pathogenesis of pre-eclampsia.     

Activation of the novel prothrombinase, fg12, as a basis for the pregnancy complications spontaneous abortion and pre-eclampsia

PROBLEM: Impaired trophoblast invasion during the first trimester of pregnancy is linked to spontaneous abortion, and defective invasion in the second trimester to hypertension + proteinuria (pre-eclampsia). Hypertension developing during the third trimester of human pregnancy represents, in part, a corrective response in the mother to provide adequate placental perfusion for fetal growth when trophoblast has not to invaded and converted the myometrial porprtion of maternal spiral arteries into to low resistance-high capacitance conduits. Deportation of vesicles from hypoxemic trophoblast is thought to cause hypertension plus proteinuria, vascular damage and a systemic coagulopathy. Trophoblast invasion may be inhibited by local cytokines, such as TGF-betas but Thl-type cytokines associated with pre-eclapmsia and spontaneous abortions (e.g., IL-1, TNF-alpha, IFN-gamma) are not known to inhibit migration at in situ concentrations. Trophoblast invasion is also inhibited by the binding of surface integrins to fibronectin and fibrin, and fibrin production is stimulated by these Th1 cytokines via up-regulation of prothrombinases(s) such as fg12 which directly and via TNF-alpha-facilitated inflamation compromise trophoblast cell integrity. We, therefore, asked if fg12 expression and TNF-alpha are increased in first trimester human miscarriage and in third trimester pre-eclampsia. METHODS: fg12 mRNA was detected using in situ hybridization and fg12 protein by immunohistochemistry. TNF-alpha mRNA and protein were similarly tested. The techniques were validated using uterine sections from day 8.5 of CBA x DBA/2 pregnancies, and then were applied to sections of placentae from normal and pre-eclamptic pregnancies with and without intrauterine fetal growth restriction (IUGR). Fibrin was detectectd by immunohistochemistry. RESULTS: Expression of fg12 protein correlated with fg12 mRNA expression in mouse uteri and in placentae from normal human pregnancies. Increased expression of fg12 and TNF-alpha mRNA and protein, and increased fibrin deposition was detected in placental trophoblast. CONCLUSIONS: Activation of fg12 prothrombinase by Th1-type cytokines in pregnancy may lead to spontaneous abortion, or in ongoing pregnancy, to pre-eclampsia and/or IUGR.     

Increased levels of pregnancy-associated plasma protein-A2 in the serum of pre-eclamptic patients

Pregnancy-associated plasma protein-A and -A2 (PAPP-A and -A2) are proteases that cleave insulin-like growth factor-binding proteins (IGFBPs), resulting in local activation of IGF signaling pathways. Here, we examined PAPP-A and -A2 mRNA and protein levels in placenta and maternal sera from women with pre-eclampsia and compared them with samples from uncomplicated pregnancy. PAPP-A2 but not PAPP-A mRNA and protein were elevated in pre-eclamptic placenta (P < 0.01). PAPP-A2 is normally produced in placental syncytiotrophoblast cells and maternal decidua. PAPP-A2 in syncytiotrophoblast cells was dramatically increased in pre-eclampsia. Maternal serum concentrations of PAPP-A2 but not PAPP-A were also significantly elevated in pre-eclampsia as compared with uncomplicated pregnancy. mRNA levels of IGFBP5, a specific substrate for PAPP-A2 protease activity, were also significantly increased, suggesting a potential role for IGFBP5 in fetal and placental growth suppression during pre-eclampsia. However, IGFBP5 protein levels were not increased in placenta from pre-eclampsia, possibly due to cleavage by up-regulated PAPP-A2. These data might imply that PAPP-A2 may be up-regulated in pre-eclamptic pregnancy to compensate for IGFBP5-mediated suppression of the IGF pathway, although final birthweights are still low in pre-eclamptic pregnancy.     

孕激素对绒毛外滋养细胞VEGF表达的影响

目的通过研究孕激素对绒毛外滋养细胞孕激素受体(PR)和血管内皮生长因子表达的影响,探讨P对绒毛外滋养细胞侵袭能力的影响及维持妊娠的机理。方法采用半定量PCR和定量PCR测定孕激素干预后绒毛外滋养细胞表达PR mRNA和VEGF mRNA的水平。结果(1)不同浓度的孕激素PRA表达量极低,PRB mRNA的表达没有显著差异(P>0.05);(2)VEGF mRNA与孕激素浓度变化无明显相关,反而与PRB mRNA的水平成反比关系。结论(1)孕激素作用的发挥取决于PR的水平,与激素本身的浓度没有明显关系;(2)PRB能下调滋养细胞分泌VEGF的能力从而抑制其侵袭力,提示PR异常可能是导致绒毛外滋养细胞侵袭能力发生改变而引起相关疾病的原因之一。     

Localization of heparanase in normal and pathological human placenta

Degradation of extracellular matrix (ECM) components is critical for invasion. Heparan sulphate proteoglycans are abundant in the ECM of the placenta and the decidua, hence their degradation may disassemble the matrix and facilitate placentation and trophoblast invasion. This study investigates the expression of heparanase in normal and pathological placentation using RT-PCR, in-situ hybridization and immunohistochemistry analysis to detect heparanase in specific cells of the placenta and at the fetal-maternal interface throughout pregnancy. Heparanase was observed in villous cytotrophoblasts (CT), syncytial trophoblasts (ST) and in intermediate trophoblast cell columns in normal first trimester, molar and ectopic pregnancies. The heparanase protein was preferentially expressed in the endothelium of fetal capillaries, and to a much lesser extent in larger fetal vessels. Extravillous trophoblasts (EVT) invading the decidua and the maternal vessels were also heparanase positive. In the second and third trimesters, villous CT remained heparanase positive whereas ST showed variable heparanase expression. EVT invading the placental implantation site were also positively stained. A similar pattern was observed in samples obtained from pre-eclamptic placentae and from placenta accreta. Our results indicate consistent expression of heparanase in normal and abnormal placenta, in small fetal vessels and in a variety of trophoblast subpopulations with different invasive potentials.     

细胞命运决定因子numb在先兆子痫胎盘组织中的表达及作用

目的探讨先兆子痫胎盘组织中细胞命运决定因子numbs表达情况及意义。方法获取正常妊娠胎盘组织以及先兆子痫胎盘组织,分别运用实时荧光定量PCR法、免疫印迹法、免疫组织化学法检测numbmRNA、蛋白分布及其表达水平。结果numb在以上两种胎盘组织中均有表达,且主要集中在细胞膜上表达。numb在先兆子痫组（实验组）的表达水平明显高于正常胎盘组（对照组）（P〈0．05）。结论numb参与的滋养细胞凋亡失调可能是先兆子痫的发病机制之一。     

S100B protein expression in the amnion and amniotic fluid in pregnancies complicated by pre-eclampsia

Our aim was to investigate the expression of S100B protein in the amnion and to assess the amniotic fluid concentration in pregnancies complicated by pre-eclampsia. Samples were obtained from women who developed pre-eclampsia (n = 7), pre-eclampsia with intrauterine growth retardation (IUGR) (n = 4), normotensive IUGR (n = 7) and gestational hypertension (n = 4) during pregnancy and healthy controls who delivered at term (n = 35). To determine the difference in the expression of S100B in the amnion, we performed immunohistochemistry, western blot analysis and RT-PCR. Using enzyme-linked immunosorbent assay (ELISA), we assessed the S100B concentration in amniotic fluid. The S100B mRNA expression in the amnion of pre-eclamptic patients and patients with pre-eclampsia with IUGR was significantly higher than that in the control. The amniotic fluid S100B protein concentration of the pre-eclampsia and normotensive IUGR cases was significantly higher than that of the control. This study shows that amnion could be a source responsible for the increased concentration of S100B in amniotic fluid. In pre-eclampsia, reactive oxygen species (ROS) are generated by oxidative stress. Some pathological conditions that develop during pregnancy and are related to hypoxic stress can affect the elevation of S100B concentration in the amnion.     

Gene regulation of neurokinin B and its receptor NK3 in late pregnancy and pre-eclampsia

Elevated circulating levels of the tachykinin, neurokinin B (NKB), have been observed in women with pre-eclampsia during the third trimester of pregnancy. Currently, the molecular mechanisms responsible for these increased levels remain unknown. To understand the molecular regulation, we have compared the differences in gene expression of the tachykinins and their receptors in control and pre-eclamptic placentae and the responses of the TAC3 gene encoding NKB to proposed physiological triggers of pre-eclampsia including hypoxia and oxidative stress using real-time quantitative PCR. We have determined the placenta to be the main site of TAC3 expression with levels 2.6-fold higher than the brain. TAC3 expression was found to be significantly higher in pre-eclamptic placenta (1.7-fold, P < 0.05) than in normal controls. No evidence was found that hypoxia and oxidative stress were responsible for increases in TAC3 expression. In rat placenta, a longitudinal study in normal late pregnancy was associated with a significant down-regulation of the NKB/NK3 ligand-receptor pair (P < 0.05). The present data suggest that the increased placental expression of TAC3 is part of the mechanism leading to the increased circulating levels of NKB in pre-eclampsia.     

Tumor necrosis factor-alpha in the placenta is not elevated in pre-eclamptic patients despite its elevation in peripheral blood

PROBLEM: Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells at the early and late stages of gestation and promotes the regulation of trophoblast growth and invasion. We evaluated whether TNF-alpha levels in the placenta and blood of pre-eclamptic women differed from those with normal pregnancies. METHOD OF STUDY: The subjects were 39 pregnant women carrying single fetuses (21 normal-pregnant and 18 pre-eclamptic patients). Their average gestational age at entry was 38-39 weeks. Peripheral blood was collected before the onset of labor and separated serum was stored at -20 degrees C. A tissue segment of the placenta was cut and frozen in liquid nitrogen immediately after delivery at -80 degrees C. The frozen placental tissue was added to phosphate-buffered saline. The tissue was fully homogenized and centrifuged. Separated supernatant was stored at -80 degrees C. TNF-alpha levels in separated serum and TNF-alpha and total protein (TP) levels in separated supernatant were measured. The presence of TNF-alpha in the placenta was evaluated by immunohistochemistry in five pre-eclamptic and five normal-pregnant patients. RESULTS: Serum TNF-alpha levels were higher in pre-eclampsia than in normal pregnancies. However, TNF-alpha/TP levels in the placenta did not differ significantly between the two groups. As for TNF-alpha immunostaining of trophoblastic cells in the placenta, it was weak in three and moderate in two of the normal pregnancies, while it was absent in two, weak in one, and moderate in two in the pre-eclampsia group. CONCLUSIONS: We demonstrated no significant increase in TNF-alpha/TP levels in the placenta in pre-eclampsia despite a significant increase in serum TNF-alpha levels. There was no strong immunostaining for TNF-alpha detected by immunohistochemistry in the pre-eclampsia group. These findings suggest that TNF-alpha in the placenta is not a key cytokine to interfere with normal trophoblast invasion into the myometrium in pre-eclampsia, and that sources other than the placenta may contribute to the elevated levels of TNF-alpha found in the circulation of pre-eclamptic patients.     

Calreticulin in human pregnancy and pre-eclampsia

Pre-eclampsia is a disorder of human pregnancy that involves pregnancy-induced maternal hypertension and proteinuria. Evidence indicates that pre-eclampsia involves widespread activation of maternal endothelial cells. Calreticulin is a ubiquitously expressed, multi-functional protein that has been shown to have both pro- and anti-inflammatory effects on cultured endothelial cells in vitro and in whole animals. In order to clarify the role of this protein in normal human pregnancy and in pre-eclampsia, this study has measured expression of calreticulin in maternal blood and in placenta in patients with pre-eclampsia and in control pregnancies. There was a significant increase (approximately 5-fold) in calreticulin in plasma in term pregnant women compared with women who were not pregnant. There was no difference, however, in calreticulin in plasma from women who were sampled at first trimester, second trimester and at term. In addition, there was a significant increase (approximately 50%) in calreticulin in plasma from pre-eclamptic women compared to controls. Calreticulin mRNA and protein expression in placenta were not changed between pre-eclampsia and control pregnancies. These novel results indicate that calreticulin is increased in peripheral maternal blood early in pregnancy and remains elevated throughout normal gestation and that there is a further increase in calreticulin in pre-eclampsia.     

Distinct expression and localization of serine protease HtrA1 in human endometrium and first-trimester placenta.

Mammalian embryos cannot survive without the placenta. Development of the human placenta requires trophoblast proliferation, differentiation, and invasion as well as highly coordinated modulation of the maternal uterus. HtrA1 is a member of the recently identified mammalian HtrA (high temperature requirement factor A) serine protease family with a high level of expression in the placenta. In this study, we examined whether HtrA1 expression (mRNA and protein) is associated with placental development in the human. HtrA1 is up-regulated in both endometrial glands and decidual cells during endometrial preparation for embryo implantation and during first-trimester pregnancy at placentation. HtrA1 expression was also detected in certain trophoblast subtypes during early pregnancy. The villous syncytiotrophoblast and cytotrophoblast showed the strongest expression while the interstitial extravillous trophoblast showed the lowest or no expression of HtrA1. The distinct distribution of HtrA1 at the maternal-trophoblast interface suggests that HtrA1 may play a role in placental development.     

Pre-term birth and severe pre-eclampsia are not associated with altered expression of HLA on human trophoblasts

PROBLEM: The unusual pattern of human leukocyte antigen (HLA) expression on human trophoblasts could play an important role in successful pregnancy outcome. To determine whether alterations in HLA expression are associated with pregnancy abnormalities we have investigated expression of these antigens on chorionic and extravillous cytotrophoblasts. METHODS: Frozen tissue sections of placenta and fetal membranes were collected after pre-term spontaneous delivery, severe pre-eclampsia pre-term Caesarean section, normal term delivery and term Caesarean section. HLA expression was analyzed by immunohistochemistry. RESULTS: We did not observe differences in the expression of HLA on chorionic and extravillous cytotrophoblasts in pregnancy abnormalities. However, we noted higher expression levels of HLA class Ia molecules in amnion epithelial cells in pre-term deliveries. Furthermore, in severe pre-eclampsia the number of extravillous cytotrophoblast islands were elevated when compared with pre-term deliveries. CONCLUSIONS: No alterations in expression of HLA class Ia, HLA-G and HLA class II on human trophoblasts in pregnancy abnormalities were seen.