Aneuploid Cells in the Human Placenta

描述(申请人提供)：在真兽类动物中，胎儿的正常发育依赖于有功能的胎盘。专门的胎盘隔间将胚胎固定在子宫上，并为胚胎维持稳定的氧气和营养供应，而其他部分则管理代谢物的处理。有两个胎盘结构促进了这些功能：浮动和锚定绒毛。对漂浮绒毛的遗传构成进行了详细的研究。关于锚定绒毛的遗传组成知之甚少，但我们对从锚定绒毛或子宫壁分离的胎儿细胞进行的细胞遗传学研究显示，意外地有很大一部分侵袭性细胞滋养细胞(CTB)携带数字染色体异常。我们推测，胎儿-母体交界处的非整倍体是正常胎盘形成程序的一个组成部分。我们假设，在没有突变的情况下，CTB的基因组变化，如染色体的获得或丢失，是限制CTB增殖和侵袭能力的正常细胞机制。为了验证我们的假设，我们建议研究CTB的核型以及沿着侵袭性CTB表型的细胞增殖或分化的表型标记。在这里提出的研究中，在早期胚胎发育、胎盘功能、分子细胞遗传学和数字图像处理方面具有互补专业知识的研究人员将合作定位不同胎盘隔室中的非整倍体细胞。首先，我们将确定在正常胎盘标本和携带21三体的胎盘组织中，子宫壁和锚定以及浮动绒毛中染色体数目异常的类型。接下来，我们将确定正常和三体中期胎盘基因组不稳定性的空间分布。使用的技术将是荧光原位杂交(FISH)、共聚焦显微镜和厚组织切片的3D图像重建，以定位组织学背景下的非整倍体细胞。最后，我们将通过BrdU掺入和人类白细胞抗原G基因的表达来确定非整倍体对CTB增殖能力的影响，这是CTB沿侵袭途径分化的重要标志。在本项目结束时，我们将开发出对间期细胞中所有24条人类染色体进行准确评分的技术，并论证了在厚组织切片中对非整倍体细胞进行三维定位的可行性。我们还将详细了解母胎交界处非整倍体细胞的频率、定位和类型，以及非整倍体对CTB增殖和HLA-G基因表达的影响。

DESCRIPTION (provided by applicant): In eutherian mammals, proper fetal development depends on a functional placenta. Specialized placental compartments anchor the embryo to the uterus and maintain a steady supply of oxygen and nutrients to the embryo, while other parts manage the disposal of metabolites. Two placental structures facilitate these functions: floating and anchoring villi. The genetic makeup of floating villi has been studied in detail. Little is known about the genetic make-up of anchoring villi, but our cytogenetic studies of fetal cells isolated from anchoring villi or the uterine wall revealed an unexpected large fraction of invading cytotrophoblasts (CTBs) that carry numerical chromosomal aberrations. We postulate that aneuploidy at the fetal-maternal interface is an integral part of the normal placentation program. We hypothesize that in the absence of mutations, genomic changes in CTBs such as gains or losses of chromosomes are normal cellular mechanisms that limit the proliferative as well as the invasive capabilities of CTBs. To test our hypothesis, we propose to investigate the karyotype of CTBs as well as phenotypic markers of cell proliferation or differentiation along an invasive CTB phenotype. In the study proposed here, investigators with complementary expertise in early embryonic development, placental function, molecular cytogenetics and digital image processing will collaborate to localize aneuploid cells in different placental compartments. First, we will determine the types of numerical chromosome abnormalities in the uterine wall and in anchoring as well as floating villi in normal placental specimens and placental tissues carrying a trisomy 21. Next, we will determine the spatial distribution of genomic instability in normal and trisomic second trimester placentas. Techniques used will be fluorescence in situ hybridization (FISH), confocal microscopy and 3D image reconstruction of thick tissue sections to localize aneuploid cells in their histological context. Finally, we will determine the effects of aneuploidy on the proliferative capacity of CTBs as measured by BrdU incorporation and expression of the HLA-G gene, an important marker for CTB differentiation along the invasive pathway. At the end of this project, we will have developed the technology to accurately score all 24 human chromosomes in interphase cells and demonstrated the feasibility to localize aneuploid cells in 3-dimension in thick tissue sections. We will also have detailed information about the frequency, localization and types of aneuploid cells at the fetal-maternal interface and the effects of aneuploidy on CTB proliferation and HLA-G gene expression.